CN117343962A - Immune compatible human pluripotent stem cell, preparation method and application thereof - Google Patents

Immune compatible human pluripotent stem cell, preparation method and application thereof Download PDF

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CN117343962A
CN117343962A CN202210752354.6A CN202210752354A CN117343962A CN 117343962 A CN117343962 A CN 117343962A CN 202210752354 A CN202210752354 A CN 202210752354A CN 117343962 A CN117343962 A CN 117343962A
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cells
hla
leu
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杨黄恬
张鹏
饶森乐
章小清
刘玲
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Tongji University
Shanghai East Hospital Tongji University Affiliated East Hospital
Shanghai Institute of Nutrition and Health of CAS
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Tongji University
Shanghai East Hospital Tongji University Affiliated East Hospital
Shanghai Institute of Nutrition and Health of CAS
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Priority to PCT/CN2023/104109 priority patent/WO2024002279A1/en
Publication of CN117343962A publication Critical patent/CN117343962A/en
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Abstract

The invention provides an immune compatible human pluripotent stem cell, a preparation method and application thereof. The modified human pluripotent stem cells can be induced to differentiate into various tissue/organ cells, have ideal immune compatibility and have wide application environments.

Description

Immune compatible human pluripotent stem cell, preparation method and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an immune compatible human pluripotent stem cell capable of escaping transplant recipient immune rejection, a preparation method and application thereof.
Background
The cell transplantation treatment is to transplant the specific powerful cells generated after the characteristics of some cells with specific functions are obtained by adopting a bioengineering method and/or are treated by in vitro amplification, special culture and the like into a patient so as to repair, supplement, replace, regulate or remove damaged or diseased cells/tissues, thereby achieving the purpose of curing diseases. Therefore, the application of the cell technology can treat various diseases, such as degenerative and injury diseases of nervous system, bone diseases, diabetes, cardiovascular and cerebrovascular diseases and the like, and has wide application prospect. However, the transplant recipients may develop immune rejection to non-self cells, making it difficult for the transplanted cells to survive in the transplant recipients for a long period of time. Even if a suitable donor tissue organ is received matching human leukocyte antigens (human leukocyte antigen, HLA), the recipient patient still needs to take immune rejection drugs for a long period of time, increasing patient burden and also presenting an infection/cancer risk, as complete HLA matching is not possible.
The essence of immune rejection is an immune response induced by alloantigens on the cell surface, mediated primarily by HLA in humans. HLA has extremely high polymorphism, and HLA matching can reduce immune rejection of transplanted cells by a transplant recipient. Based on the mechanism of immune rejection, the following methods exist to alleviate immune rejection:
1. HLA matching. Mainly used in organ and hematopoietic stem cell transplantation, and can reduce rejection by HLA matching of donor/receptor. However, due to the scarcity of suitable donors and the extremely high polymorphism of HLA, finding suitable donors is difficult, and it is difficult to meet the needs of organ transplant patients.
2. Autologous induced pluripotent stem cells (induced pluripotent stem cells, iPSCs). iPSCs are derived from reprogramming of cells from the body, possess HLA genes that are fully identical to the donor, and theoretically iPSCs and their derived cells can also escape autoimmune rejection. However, autologous iPSCs and derived cells thereof have long preparation cycle, difficult quality control, high cost, larger application difficulty in diseases with strong timeliness and narrow treatment time window, and are very limited in use. Secondly, for patients with genetic diseases, the iPSCs source cells of the patients have abnormal functions, so that potential safety hazards are brought. In addition, various degenerative and damaging diseases are well developed in people of high ages, such as myocardial infarction, parkinson's disease and the like, and the difficulty in establishing iPSCs is high for the people.
3. And establishing an HLA matching iPSCs library which can cover most people. Because of the extremely high polymorphism of HLA, the cost for establishing and maintaining the iPSCs ligand library is extremely high, the period is very long, and the iPSCs ligand library is still difficult to apply in a short time. Moreover, there are also differences between tissue cells derived from different iPSCs cell lines, such as cardiomyocytes, and the use of non-uniformized cells may adversely affect subsequent treatments.
All of the above methods have the disadvantages of difficult acquisition, high cost and difficult quality control, so that the establishment of universal low-immunogenicity donor cells is an urgent need in the art.
Through the search of the documents and patents published and published at present, the technical routes for obtaining the general cell are as follows: (1) By knocking out a key HLA molecule on a chromosome, constructing pseudo HLA homozygote cells, and increasing universality of the obtained cells; (2) By knocking out HLA I family HLA-A, -B, -C and HLA II family molecules and assisting in over-expressing don' T-eat me signal CD47 molecules, membrane positioning HLAG molecules and T cell inhibitor PD-L1, and escaping immune cell recognition and killing effects; (3) By completely knocking out HLA I family and HLA II family molecules and assisting in the overexpression of a "don' T eat me" signal CD47 molecule, the recognition and killing effect of the escape T cells and macrophages are realized; (4) Recognition and killing of cytotoxic T cells and natural killer cells (NK cells) is escaped by knocking out classical HLA I molecules mediating antigen presentation and overexpressing HLA-G1 and secretory HLA-G5 molecules with immunosuppressive effects, as in the national patent application filed by co-inventors Zhang Xiaoqing et al (CN 113528448 a) in the present invention. Compared with the 4 technical routes, the (1) th method has extremely high difficulty, requires precise control of single alleles of a plurality of HLA molecules, and increases the risk of mutation of the genome; the (2) needs to cause a large range (greater than 13 kb) of genome deletions to achieve knockout-A, -B, -C results, bringing potential instability to the genome; the immunosuppressive signal of the (3) selection is single, the main acting target is macrophage, compared with HLAG with broad-spectrum immunosuppressive effect, the better selection is possible. Different immunosuppressant factors have different acting objects and different acting effects, so that the selection of the immunosuppressant factors is the key of different technical routes. The co-inventors of the present invention constructed a B2Mm/sHLAG embryonic stem cell (human embryonic stem cells, hESCs) line using simultaneous over-expression of membrane-localized HLA-G1 and soluble HLA-G5, reducing the immunogenicity of human embryonic stem cells, wherein embryonic stem cells were studied without involvement of hPSCs and yet to be further expanded in their application environment.
Disclosure of Invention
The invention provides an immune compatible human pluripotent stem cell, a preparation method and application thereof, and the immune compatible human pluripotent stem cell has wider application.
In a first aspect of the invention, there is provided a method of preparing an immunocompatible human pluripotent stem cell comprising engineering the human pluripotent stem cell to:
(a) Free B2M protein is not expressed, HLA-G1 and secretory HLA-G5 are expressed (including over-expression); and (b) does not express CIITA protein.
In one or more embodiments, in (a), the genome of the human pluripotent stem cell is modified, and the polynucleotide encoding HLA-G1 is fused to a B2M gene endogenous to the human pluripotent stem cell, thereby expressing the B2M-HLA-G1 fusion protein and not expressing the free B2M protein; preferably, the HLA-G1 encoding gene is introduced into the endogenous B2M gene at a position before the stop codon in exon 3, or the stop codon in exon 3 of the endogenous B2M gene is replaced with the HLA-G1 encoding gene; more preferably, the modification is performed by a gene editing method.
In one or more embodiments, in the (a), introducing into a human pluripotent stem cell an exogenous polynucleotide encoding a B2M-HLA-G5 fusion protein comprising B2M and HLA-G5; preferably, the exogenous polynucleotide encoding a B2M-HLA-G5 fusion protein is introduced using a recombinant vector (e.g., a viral vector, more particularly a lentiviral vector).
In one or more embodiments, the B2M-HLA-G1 fusion protein further comprises a flexible connecting peptide between B2M and HLA-G1, preferably a flexible connecting peptide as shown in SEQ ID NO. 3; and/or, the B2M-HLA-G1 fusion protein comprises the following components from the N end to the C end in sequence: B2M and HLA-G1; preferably, the amino acid sequence of the B2M-HLA-G1 fusion protein is shown as SEQ ID NO. 4; more preferably, the nucleic acid sequence of the B2M-HLA-G1 fusion protein is shown as SEQ ID NO. 9.
In one or more embodiments, the B2M-HLA-G5 fusion protein further comprises a flexible connecting peptide between B2M and HLA-G5, preferably a flexible connecting peptide as shown in SEQ ID NO. 3; and/or, the B2M-HLA-G5 fusion protein comprises the following components from the N end to the C end in sequence: B2M and HLA-G5; preferably, the amino acid sequence of the B2M-HLA-G5 fusion protein is shown as SEQ ID NO. 8; more preferably, the nucleic acid sequence of the B2M-HLA-G5 fusion protein is shown as SEQ ID NO. 10.
In one or more embodiments, in (b), the knockout is made for exon 3 of the CIITA gene in the human pluripotent stem cell genome; preferably, the knockout is performed by a gene editing method; more preferably, the gene editing is performed with gRNA (GGGAGGCTTATGCCAATAT) of the nucleotide sequence shown in SEQ ID NO. 11.
In one or more embodiments, the method of preparation includes one or more of the following A-D:
A. the HLA-G1 comprises: a polypeptide with an amino acid sequence shown as SEQ ID NO. 1; or, a derivative polypeptide (including an active fragment, an active variant) having an amino acid sequence having 90% or more sequence identity to SEQ ID NO. 1 and having the function of the polypeptide shown in SEQ ID NO. 1;
B. the B2M includes: a polypeptide with an amino acid sequence shown as SEQ ID NO. 2; or, a derivative polypeptide (including an active fragment, an active variant) having an amino acid sequence having 90% or more sequence identity to SEQ ID NO. 2 and having the function of the polypeptide shown in SEQ ID NO. 2;
C. the CIITA protein comprises: a polypeptide with an amino acid sequence shown as SEQ ID NO. 5; or, a derivative polypeptide (including an active fragment, an active variant) having an amino acid sequence having 90% or more sequence identity to SEQ ID NO. 5 and having the function of the polypeptide shown in SEQ ID NO. 5;
D. the HLA-G5 comprises: a polypeptide with an amino acid sequence shown as SEQ ID NO. 6; or a polypeptide fragment having an amino acid sequence having 90% or more sequence identity to SEQ ID NO. 6 and having the function of the polypeptide shown in SEQ ID NO. 6.
In one or more embodiments, the "90% or more sequence identity" includes: more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% sequence identity.
In a second aspect of the present invention, there is provided an immune-compatible human pluripotent stem cell which (a) does not express free B2M protein, expresses HLA-G1 and secretory HLA-G5; and (b) does not express CIITA protein. As a preferred embodiment or embodiments of the present invention, the human pluripotent stem cells: the endogenous B2M gene in the genome is fused with a polynucleotide encoding HLA-G1; comprising an exogenous polynucleotide encoding a B2M-HLA-G5 fusion protein; and, the CIITA gene is knocked out in the genome.
In one or more embodiments, the immune-compatible human pluripotent stem cells are constructed by the methods of preparing immune-compatible human pluripotent stem cells described herein.
In a third aspect of the invention there is provided the use of an immunocompatible human pluripotent stem cell for the preparation of cells suitable for transplantation by induced differentiation; preferably, the cells suitable for transplantation are tissue or organ cells; more preferably, the tissue or organ cells include (but are not limited to): cardiovascular precursor cells, cardiomyocytes, endothelial cells, smooth muscle cells, neural cells, hematopoietic stem cells, myeloid cells (including granulocytes, monocytes, macrophages, erythrocytes, platelets), gonomic cells (including natural killer cells, T cells, B cells), retinal pigment epithelial cells, islet B cells, liver cells (including hepatocytes, cholangiocytes, liver endothelial cells, hepatic stellate cells, kupffer cells, mesothelial cells), keratinocytes, skeletal muscle cells, adipocytes, bone cells, chondrocytes, mesenchymal stem cells.
In a fourth aspect of the invention, there is provided a method of preparing cells suitable for transplantation comprising: (a) The immune-compatible human pluripotent stem cells are prepared and obtained by the method for preparing the immune-compatible human pluripotent stem cells; (b) Further inducing differentiation of the cells of (a) to obtain cells suitable for transplantation; preferably, the cells suitable for transplantation are tissue or organ cells; more preferably, the tissue or organ cells include (but are not limited to): cardiovascular precursor cells, cardiomyocytes, endothelial cells, smooth muscle cells, neural cells, hematopoietic stem cells, myeloid cells (including granulocytes, monocytes, macrophages, erythrocytes, platelets), gonomic cells (including natural killer cells, T cells, B cells), retinal pigment epithelial cells, islet B cells, liver cells (including hepatocytes, cholangiocytes, liver endothelial cells, hepatic stellate cells, kupffer cells, mesothelial cells), keratinocytes, skeletal muscle cells, adipocytes, bone cells, chondrocytes, mesenchymal stem cells.
In one or more embodiments, the method of preparing cells suitable for transplantation, wherein the tissue or organ cells are cardiomyocytes, are prepared by the method of CHIR99021 followed by IWR-1 induction; or the tissue or organ cells are endothelial cells, and are prepared by adopting a method of induction of CHIR99021, bFGF and VEGF+BMP4 in sequence.
In one or more embodiments, the CHIR99021, IWR-1, bFGF, VEGF, or BMP4 further comprises their cognate molecules.
In one or more embodiments, the CHIR99021 is a final concentration of 6 μm that can float up and down within 50%, preferably within 40%, preferably within 30%, preferably within 20%, preferably within 10%. Preferably, the CHIR99021 is treated for 2±0.5 days.
In one or more embodiments, the IWR-1 is at a final concentration of 5 μM, which may float up and down within 50%, preferably within 40%, preferably within 30%, preferably within 20%, preferably within 10%. Preferably, the IWR-1 is treated for 2+ -0.5 days.
In one or more embodiments, the bFGF is at a final concentration of 50ng/ml that floats up and down within 50%, preferably within 40%, preferably within 30%, preferably within 20%, preferably within 10%. Preferably, the bFGF is treated for 1±0.25 days.
In one or more embodiments, the VEGF is at a final concentration of 50ng/ml, which can float up and down within 50%, preferably within 40%, preferably within 30%, preferably within 20%, preferably within 10%.
In one or more embodiments, the BMP4 is at a final concentration of 50ng/ml, which can float up and down within 50%, preferably within 40%, preferably within 30%, preferably within 20%, preferably within 10%.
In one or more embodiments, the vegf+bmp4 is treated for 2±0.5 days.
In a fifth aspect of the present invention, there is provided a cardiomyocyte or endothelial cell, an immune-compatible human pluripotent stem cell prepared by the method for preparing an immune-compatible human pluripotent stem cell according to the invention or derived from an immune-compatible human pluripotent stem cell according to the invention.
In a sixth aspect of the invention there is provided the use of said cardiomyocytes in the manufacture of a composition or medicament for the treatment of a heart-related disease or disorder; preferably, the heart-related disease or disorder is myocardial injury, myocardial infarction, myocardial ischemia, ischemia reperfusion injury, or other heart injury; more preferably, the heart-related disease or disorder is ischemia/reperfusion injury.
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
Drawings
FIG. 1, construction of immunocompatible cells and strategy for obtaining.
FIG. 2, construction of immunocompatible cells and strategy for obtaining. A, designing gRNA at the Exon2 position of B2M and the Exon3 position of CIITA, co-transfecting gRNA expression plasmid and Cas9 expression plasmid into hPSCs, then picking monoclonal, and carrying out PCR sequencing verification. Clones with frameshift mutations were B2M and CIITA double knockout clones. B, firstly constructing B2Mm/sHLAG hPSCs (see CN 113528448A), then co-transfecting gRNA and Cas9 plasmid, selecting monoclonal, performing PCR sequencing verification, and cloning the clone with frame shift mutation as CIITA -/- B2Mm/sHLAG-hPSCs。
FIG. 3 flow cytometry identification of wt-hPSCs, DKO-hPSCs and CIITA -/- Expression of the pluripotency markers OCT4 and SSEA4 in B2Mm/sHLAG-hPSCs cells. A, flow cytometry identified OCT4 expression (left) and OCT4 positive percentage (right). B, flow cytometry identified SSEA4 expression (left) and OCT4 positive percentage (right). n=3.
FIG. 4 immunofluorescence identification of wt-hPSCs, DKO-hPSCs and CIITA -/- Expression of the pluripotency markers OCT4 and SOX2 of B2Mm/sHLAG-hPSCs cells. Ruler: 20 μm.
FIG. 5, HLA family molecule expression identification. A, the expression of HLA-a/B/C, a representative molecule of HLA I, is identified in a flow-through manner. B, identifying HLA-G1 molecule expression in a flow mode. C, flowthrough identifies the extent of HLA-DR enrichment on cell membranes, n=3. D, immunoblotting identified the expression of secretory HLA-G5 pairs in the cell supernatant. * p <0.05.
FIG. 6, identification of differentiated endothelial cells and cardiomyocytes. And A, identifying the morphology of the differentiated endothelial cells and the purity of the endothelial cells. Purity identification the endothelial marker CD144 expression proportion was identified by flow cytometry. Scale bar = 100 μm. n=3. And B, identifying the morphology and purity of the differentiated myocardial cells. Purity identification the expression proportion of the cardiomyocyte marker cTNT was identified by flow cytometry. Scale bar = 100 μm. n=3. Immunofluorescence identified the expression of sarcomere alpha-ACTININ and gap protein Connexin-43 in differentiated cardiomyocytes. Scale bar = 10 μm.
FIG. 7 immune cell pair CIITA -/- Identification and killing of B2 Mm/sHLAG-hPSCs-derived cardiomyocytes. Flow cytometry analysis of wt-CMs, CIITA -/- B2Mm/sHLAG-CMs were co-cultured with hBMCs for the effect of early activation of T cells in hBMCs (A), for the effect of activation of T cell proliferation (B), for the effect of killing of co-cultured cardiomyocytes by hBMCs (C) and for the secretion of IFN-gamma (D), respectively. NK-92 and DKO-CMs or CIITA -/- Killing effect of B2Mm/sHLAG-CMs on myocardial cells (E) and secretion amount of IFN-gamma of NK-92 cells (F). n=5 (a); n=4 (B); n=5 (C); n=5 (D); n=6 (E); n=5 (F). * P is p<0.05,***p<0.001。
FIG. 8 evaluation of cell retention after transplantation of hPSCs-derived cardiomyocytes after myocardial infarction of humanized mice. A, a research schematic. B, day 28 post-implantation cardiac slice immunofluorescence. alpha-ACTININ: myofilament structural proteins. hu-KU80: human KU80 protein, antibodies used herein bind only to human KU80 protein and do not cross-react with mouse KU 80. Scale bar = 500 μm. C, cell retention statistics (calculated as area). Female mice, n=3-7; male mouse, n=3. D, cell retention statistics (calculated by qRT-PCR), female mice, n=4-5; male mouse, n=3. * p <0.05, < p <0.01, < p <0.001.
FIG. 9, hu-mice myocardial infarction heart re-muscle assessment. A, day 28 post-implantation mice heart sections were stained with Masson. Scale bar = 1mm. And B, counting the re-muscle proportion of the heart of the mice. Female mice, n=3-7; male mouse, n=3. * p <0.05, < p <0.01, < p <0.001.
Detailed Description
The present inventors have found that in the course of using B2Mm/sHLAG hESCs, there is still a need for improvement in immune compatibility. The B2Mm/sHLAG hESCs perform better when they are mainly used for cell therapy of degenerative diseases. But when it is in some other transplantation environment, there is still a phenomenon in which immune cells (including antigen presenting cells) are activated. For example, when the present inventors applied it to cell therapy of acute injury, such as high inflammation level at the infarcted heart injury site. Clinically, transplanted exogenous cells are required to be implanted into a lesion or to play a therapeutic role near the lesion, and damaged tissues often accompany excessive activation of inflammatory reactions at lesions, so that the levels of tissue cytokines at lesions are higher. When hescs are transplanted to induce differentiated tissue/organ cells, immune rejection response against such cells is enhanced. After extensive experimental studies, analyzing a large number of molecules that may be related to such phenomena, the inventors determined targets for further engineering. Based on this, the invention also discloses a further modified immune compatible cell which has wider application environment.
Terminology
The term "pluripotent stem cells (pluripotent stem cells, PSCs)" refers to cells that are capable of self-renewal and proliferation while remaining in an undifferentiated state and that can be induced to differentiate into specialized cell types under appropriate conditions. As used herein, "pluripotent stem cells" include pluripotent stem cells and other types of stem cells, including embryonic, amniotic or somatic stem cells. As used herein, human pluripotent stem cells (hPSCs) may be human embryonic stem cells (human embryonic stem cells, hESCs) isolated or obtained using human embryos within 14 days of fertilization that have not undergone in vivo development. Exemplary human stem cell lines include H9 human pluripotent stem cell lines, which may be commercially available H9 hESC cell lines, for example. Additional exemplary stem cell lines include those obtainable by the collection of National Institutes of Health Human Embryonic Stem Cell Registry and Howard Hughes Medical Institute HUES (as described by Cowan, c.a. et al, new England j.med.350:13 (2004)).
As used herein, a "pluripotent stem cell" has the potential to differentiate into any of the following three germ layers: endoderm (e.g., gastric junctions, gastrointestinal tract, lungs, etc.), mesoderm (e.g., muscle, bone, blood, genitourinary tissue, etc.), or ectoderm (e.g., epidermal tissue and nervous system tissue). The term "pluripotent stem cell" as used herein also includes "induced pluripotent stem cells", "iPS", "iPSC" or "iPSCs", a pluripotent stem cell derived from a non-pluripotent cell. Examples of parent cells include somatic cells that have been reprogrammed by various means to induce an induced pluripotent, undifferentiated phenotype. Such "iPS", "iPSC" or "iPSCs" cells may be produced by inducing the expression of certain regulatory genes or by exogenous application of certain proteins. Methods of inducing iPS cells are known in the art. As used herein, "hiPSC," "hPSC," or "hPSCs" are human induced pluripotent stem cells, "miPSC," "miPSCs," or "mPSCs" are murine induced pluripotent stem cells.
The term "pluripotent stem cell characteristics" refers to cellular characteristics that distinguish pluripotent stem cells from other cells. The ability to produce offspring capable of differentiating under appropriate conditions into cell types that together display characteristics related to cell lineages from all three germinal layers (endoderm, mesoderm and ectoderm) is a pluripotent stem cell characteristic. The expression or non-expression of certain combinations of molecular markers is also a pluripotent stem cell characteristic. For example, human pluripotent stem cells express at least several and in some embodiments all markers from the following non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, sox2, E-cadherin, UTF-1, oct4, rex1 and Nanog. The cell morphology associated with pluripotent stem cells is also a characteristic of pluripotent stem cells. As described herein, cells need not be reprogrammed to endodermal progenitor cells and/or hepatocytes by pluripotency.
The term "multipotent" or "multipotent cell" refers to a cell type that can produce a limited number of other specific cell types. For example, induced pluripotent cells are capable of forming endodermal cells. In addition, pluripotent blood stem cells can differentiate themselves into several types of blood cells, including lymphocytes, monocytes, neutrophils, and the like. The term "oligopotent" refers to the ability of an adult stem cell to differentiate into only a few different cell types. For example, lymphoid or myeloid stem cells are capable of forming cells of the lymphoid or myeloid lineage, respectively. The term "monoenergetic" refers to the ability of a cell to form a single cell type. For example, spermatogonial stem cells can only form sperm cells.
The term "non-pluripotent cell" refers to a mammalian cell that is not a pluripotent cell. Examples of such cells include differentiated cells and progenitor cells. Examples of differentiated cells include, but are not limited to, cells from tissue selected from the group consisting of bone marrow, skin, skeletal muscle, adipose tissue, and peripheral blood. Exemplary cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T cells. The starter cells used to produce induced pluripotent cells, endodermal progenitor cells, and hepatocytes may be non-pluripotent cells. Differentiated cells include, but are not limited to, pluripotent cells, oligopotent cells, monopotent cells, progenitor cells, and terminally differentiated cells. In certain embodiments, lower energy cells are considered "differentiated" relative to more energy cells.
The term "somatic cell" is a cell that forms an organism. Somatic cells include cells that constitute organs, skin, blood, bone, and connective tissue in the living body, but do not include germ cells. The cells may be derived from, for example, a human or non-human mammal. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cows, and non-human primates. In some embodiments, the cell is from an adult or non-human mammal. In some embodiments, the cells are from neonates, adults, or non-human mammals.
As used herein, the term "subject" or "patient" refers to any animal, such as a domestic animal, zoo animal, or human. The "subject" or "patient" may be a mammal, such as a dog, cat, bird, livestock, and also includes humans. Specific examples of "subjects" and "patients" include, but are not limited to, individuals (particularly humans) having diseases or disorders associated with liver, heart, lung, kidney, pancreas, brain, neural tissue, blood, bone marrow, and the like. Mammalian cells may be from human or non-human mammals. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cattle, and non-human primates (e.g., chimpanzees, macaques, and apes).
The term "HLA" or "human leukocyte antigen" complex is a complex of genes encoding human Major Histocompatibility Complex (MHC) proteins. These cell surface proteins constituting the HLA complex are responsible for modulating the immune response to the antigen. In humans, there are two HLA: class I and class II, "HLA I" and "HLA II". HLA I includes at least three proteins, HLA-A, HLA-B and HLA-C, which present peptides from within cells. HLA II comprises at least five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ and HLA-DR, which present antigens from outside the cell to T lymphocytes. It will be appreciated that the use of "MHC" or "HLA" is not meant to be limiting, as it depends on whether the gene is from a Human (HLA) or a Murine (MHC). Thus, these terms are used interchangeably herein when referring to mammalian cells.
The term "gene knockout" refers to a process of rendering a particular gene inactive in the host cell in which it is located, which results in the production of no protein of interest or inactive form. As will be appreciated by those skilled in the art and further described below, this can be accomplished in a number of different ways, including removal of all or part of the nucleic acid sequence from the gene, or disruption of the sequence with other sequences, removal or alteration of regulatory components (e.g., promoters) such that the gene is not transcribed, alteration of reading frames, prevention of translation by binding to mRNA, alteration of regulatory components of the nucleic acid, and the like. For example, all or part of the coding region of the gene of interest may be deleted or replaced with a "nonsense" sequence, all or part of the regulatory sequence (e.g., promoter) may be deleted or replaced, the translation initiation sequence may be deleted or replaced, etc. Typically, the knockout is performed at the genomic DNA level such that the progeny of the cell also permanently carry the knockout.
The term "gene knock-in/introduction" refers to the process of adding genetic functions to a host cell. This results in increased levels of encoded protein. As will be appreciated by those skilled in the art, this may be accomplished in several ways, including adding one or more additional copies of the gene to the host cell or altering regulatory components of the endogenous gene, thereby increasing the expression of the protein. This can be achieved by modifying the promoter, adding a different promoter, adding an enhancer or modifying other gene expression sequences, typically knock-in techniques result in the integration of additional copies of the transgene into the host cell.
The term "beta-2 microglobulin" or "beta 2M" or "B2M" protein may refer to a protein having the following SEQ ID NO:2, or an amino acid sequence as set forth in SEQ ID NO:2 and having a similar biological activity to the human β2m protein having more than 90% sequence identity to the amino acid sequence shown in seq id no. The term "CIITA" protein may refer to a polypeptide having the following SEQ ID NO:5, or an amino acid sequence as set forth in SEQ ID NO:5 and having a biological activity similar thereto.
In the context of cells, "wild-type" refers to cells found in nature. However, in the context of pluripotent stem cells, as used herein, it is also meant to encompass iPSCs that may contain nucleic acid changes that result in pluripotency, but that do not undergo the gene editing program of the present invention to achieve low immunogenicity.
The term "allogeneic" refers to a genetic difference between a host organism and cell transplantation in which an immune response is generated.
"B2M" herein -/- "means that a diploid cell has an inactivated B2M gene in both chromosomes, which can be accomplished in various ways. Similarly, "CIITA" is herein -/- "refers to a diploid cell having inactivated CIITA genes in both chromosomes. This may be accomplished in a variety of ways, as described herein.
As used herein, "m/sHLAG" is the abbreviation for membrane (m) localization HLA-G1 with soluble(s) HLA-G5.
As used herein, a "B2Mm/sHLAG hPSCs" cell line is a cell line that overexpresses both membrane-localized HLA-G1 and soluble HLA-G5.
As used herein, the term "expression" includes "over-expression", "recombinant expression". The "overexpression" is, for example, a significant increase in the expression level compared to the expression level in the wild-type, e.g., an increase of more than 1.2, 1.5, 2, 3, 5, 8, 10, 15, 20, 30, 50-fold or higher in the wild-type.
As used herein, "non-expressed" is relative, and also encompasses "low-expressed," "very low-expressed," e.g., the expression of an engineered cell is reduced to less than 15%, less than 10%, less than 8%, less than 5%, less than 3%, less than 2%, less than 1% or less of wild-type compared to the amount expressed in wild-type.
As used herein, the term "comprising" means that the various ingredients may be applied together in a mixture or composition of the invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "containing. As used herein, the term "effective amount" or "effective dose" refers to an amount that is functional or active in a subject and acceptable to humans and/or animals.
As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in a subject without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
Each maximum numerical limitation given in this specification includes each lower numerical limitation as if such lower numerical limitation were explicitly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Each numerical range recited in this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
Cells of the invention and uses thereof
The present invention provides a human pluripotent stem cell that avoids host immune response due to the genetic manipulation of the present invention. Specifically, the genome of the human pluripotent stem cell is engineered to: free B2M protein is not expressed, CIITA protein is not expressed, but HLA-G1 (exogenous) and secretory HLA-G5 are expressed. It will be appreciated by those skilled in the art that human pluripotent stem cells constructed by the construction method described herein are also within the scope of the present invention.
The human pluripotent stem cells allow for the derivation of "off-the-shelf" cell products for the production of specific tissues and organs. The ability to use derivatives of the human pluripotent stem cells in human patients to provide significant benefits, including the ability to avoid long-term adjuvant immunosuppressive therapy and drug use commonly seen in transplantation, e.g., the cell-derived cardiomyocytes can avoid recognition and killing of them by human immune cells with better residence in a model of cardiac ischemia/reperfusion injury. It can also provide significant cost savings because cell therapy can be used without requiring separate treatments for each patient. Thus, the human pluripotent stem cells are immunocompatible (avoid host immune responses), can be used in a broader patient population, and can be used as a universal cell source for producing universally accepted derivatives.
The present invention provides a human pluripotent stem cell that avoids host immune responses due to several genetic manipulations described in the present invention. The cells lack the primary immune antigen that elicits an immune response and are engineered to escape recognition and killing of immune cells.
Specifically, the human pluripotent stem cells of the invention do not express free B2M protein and CIITA protein, but as a preferred mode, they can successfully express secretory B2M-HLA-G5 fusion proteins. In some embodiments, the human pluripotent stem cells of the invention are achieved by integrating a recombinant nucleic acid in the genome of the human pluripotent stem cells.
Exemplary genetically manipulated techniques include homologous recombination, knock-in, ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases), CRISPR (clustered regularly interspaced short palindromic repeats)/Cas 9, and other site-specific nuclease techniques. There are a number of CRISPR/Cas9 based techniques, see for example Doudna and Charpentier, science doi 10.1126/science.1258096, which are incorporated herein by reference. These techniques enable double-stranded DNA breaks at the desired locus. These controlled double strand breaks promote homologous recombination at specific locus sites. This process focuses on targeting specific sequences of nucleic acid molecules, such as chromosomes, with endonucleases that recognize and bind sequences and induce double strand breaks in nucleic acid molecules. Double strand breaks are repaired by error-prone non-homologous end joining (NHEJ) or by Homologous Recombination (HR).
As will be appreciated by those skilled in the art, many different techniques can be used to engineer the pluripotent stem cells of the invention, as well as to make the human pluripotent stem cells deficient in the primary immune antigen that elicits an immune response, as described herein, and engineered to escape recognition and killing of immune cells.
In general, these techniques may be used alone or in combination. For example, in the construction of human pluripotent stem cells, CRISPR can be used to reduce expression of active B2M and/or CIITA proteins in engineered cells and to achieve stable transduction of genes using viral techniques (e.g., lentiviruses). Furthermore, although one embodiment sequentially knocks out B2M using CRISPR/Cas9 technology and then CIITA by CRISPR/Cas9 technology, as will be appreciated by those skilled in the art, these genes may be operated in a different order using different technologies.
For all of these techniques, well-known recombinant techniques are used to produce recombinant nucleic acids as described herein. In certain embodiments, a recombinant nucleic acid (encoding a desired polypeptide, e.g., a B2M-HLA-G1 fusion protein or CIITA protein) may be operably linked to one or more regulatory nucleotide sequences in an expression construct. The regulatory nucleotide sequences are generally suitable for the host cell and the subject to be treated. Various types of suitable expression vectors and suitable regulatory sequences are known in the art for use in a variety of host cells. In general, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art are also contemplated. The promoter may be a naturally occurring promoter, or a hybrid promoter combining elements of more than one promoter. The expression construct may be present in the cell on an episome (e.g., a plasmid), or the expression construct may be inserted into a chromosome. In a specific embodiment, the expression vector includes a selectable marker gene to allow selection of transformed host cells. Certain embodiments include expression vectors comprising a nucleotide sequence encoding a variant polypeptide operably linked to at least one regulatory sequence. Regulatory sequences for use herein include promoters, enhancers and other expression control elements. In certain embodiments, the expression vector is designed for the selection of the host cell to be transformed, the particular variant polypeptide desired to be expressed, the copy number of the vector, the ability to control that copy number, or the expression of any other protein encoded by the vector, such as an antibiotic marker.
In the present invention, cardiomyocytes derived from said human pluripotent stem cells are also provided, which are then transplanted into a patient in need thereof, thereby avoiding recognition and killing thereof by human immune cells, as well as the use of said derived cardiomyocytes in the manufacture of a composition or medicament for the treatment of heart-related diseases or disorders.
As will be appreciated by those skilled in the art, human pluripotent stem cells allow for the derivation of "off-the-shelf" cell products for the production of specific tissues and organs. The ability to use derivatives of the human pluripotent stem cells in human patients to provide significant benefits, including the ability to avoid long-term adjuvant immunosuppressive therapy and drug use commonly seen in transplantation, e.g., the cell-derived cardiomyocytes can avoid recognition and killing of them by human immune cells with better residence in a model of cardiac ischemia/reperfusion injury. It can also provide significant cost savings because cell therapy can be used without requiring separate treatments for each patient. Thus, the human pluripotent stem cells are immunocompatible and can be used in a wider population of patients as a universal cell source for producing commonly accepted derivatives.
The present invention also provides a human pluripotent stem cell having good immune compatibility (avoiding host immune response) due to several genetic manipulations described in the present invention. The cells lack the primary immune antigen that elicits an immune response and are engineered to have better immune compatibility (avoid host immune responses). It will be appreciated by those skilled in the art that human pluripotent stem cells constructed by the construction method described herein are also within the scope of the present invention.
Providing cardiomyocytes derived from said human pluripotent stem cells and then transplanting them into a patient in need thereof, thereby avoiding recognition and killing thereof by human immune cells, and also providing use of said derived cardiomyocytes in the manufacture of a composition or medicament for treating a heart-related disease or disorder, preferably, myocardial injury, myocardial infarction, myocardial ischemia, ischemia reperfusion injury, or other cardiac injury; more preferably, the heart-related disease or disorder is ischemia/reperfusion injury. The invention also provides application of the human pluripotent stem cell-derived myocardial cells in a composition or a medicament for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction and protecting myocardial ischemia injury.
In a preferred embodiment, the heart-related disease or disorder is myocardial injury, myocardial infarction, myocardial ischemia, ischemia reperfusion injury, or other cardiac injury; more preferably, the heart-related disease or disorder is ischemia/reperfusion injury. The invention also provides application of the human pluripotent stem cell-derived myocardial cells in a composition or a medicament for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction and protecting myocardial ischemia injury.
Viewed from another aspect the invention provides cardiomyocytes derived from said human pluripotent stem cells which are then transplanted into a patient in need thereof to avoid recognition and killing thereof by human immune cells, and the use of said human pluripotent stem cell derived cardiomyocytes in the manufacture of a composition or medicament for treating a heart-related disease or disorder. As a preferred mode of the invention, the heart-related disease or condition is myocardial injury, myocardial infarction, myocardial ischemia, ischemia reperfusion injury, or other cardiac injury. As a more preferred mode of the invention, the heart-related disease or condition is ischemia/reperfusion injury. It can also be said that the invention provides the application of the human pluripotent stem cell-derived cardiomyocyte in preparing a composition or a medicament for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction and protecting myocardial ischemia injury.
From a therapeutic point of view, it can also be said that the present invention provides cardiomyocytes derived from said human pluripotent stem cells for use in the treatment of heart related diseases or disorders. For example, for the treatment of diseases or conditions such as myocardial injury, myocardial infarction, myocardial ischemia, ischemia reperfusion injury, and the like. As a preferred mode of the invention, the invention provides the human pluripotent stem cell-derived cardiomyocyte for improving myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction and protecting myocardial ischemia injury.
The invention also provides a composition comprising an effective amount of said derivatized cardiomyocytes, and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In general, the pharmaceutical formulations should be compatible with the mode of administration, and the pharmaceutical compositions of the present invention may be formulated as injectable formulations, for example, using physiological saline or aqueous solutions containing glucose and other adjuvants, by conventional methods. The pharmaceutical compositions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The composition of the invention can be directly used for promoting the myocardial repair after myocardial infarction, improving the heart function after myocardial infarction and protecting myocardial ischemia injury. In addition, it may be used in combination with other therapeutic agents or adjuvants.
Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8.
The effective amount of the derivatized cardiomyocytes described herein can vary depending on the mode of administration, the severity of the disease to be treated, and the like. The selection of the preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. The mode of administration of the derivatized cardiomyocytes of the invention is not particularly limited and may be systemic or local. For example, the derivatized cardiomyocytes of the invention may be administered by means of local tissue injection, preferably myocardial injection. In addition, other modes of injection are possible, such as modes of administration to a subject including, but not limited to, intraperitoneal injection, intravenous injection, oral administration, subcutaneous injection, spinal myelin injection, intradermal injection, and the like.
The method of the invention
The invention also provides a construction method of the human pluripotent stem cells, comprising the following steps: modifying human pluripotent stem cells to: free B2M protein is not expressed, HLA-G1 and secretory HLA-G5 are expressed; and does not express CIITA protein.
In the construction method provided by the invention, the coding gene of the HLA-G1 fragment can comprise a coding sequence of a heavy chain reading frame of HLA-G1, and the HLA-G1 fragment can comprise:
a) A polypeptide fragment with an amino acid sequence shown as SEQ ID NO. 1; or alternatively, the first and second heat exchangers may be,
b) A polypeptide fragment having an amino acid sequence which has more than 90% sequence identity to SEQ ID NO. 1 and which has the function of the polypeptide fragment defined under a).
Amino acid sequence of HLA-G1 heavy chain sequence (HLA-G1 fragment):
GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD*(SEQ ID NO:1)
specifically, the amino acid sequence in b) specifically refers to: the amino acid sequence shown in SEQ ID NO. 1 is obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids at the N-terminal and/or C-terminal, and has the function of the polypeptide fragment shown in SEQ ID NO. 1. For example, the HLA-G1 fragment typically has an intact alpha heavy chain structure, which, unlike classical HLA I molecules, has predominantly immunosuppressive functions (e.g., can bind to inhibitory receptors, including predominantly ILT2/CD85j/LILRB1, ILT4/CD85d/LILRB2, and KIR2DL4/CD158d, etc., and thus can modulate B-cell, T-cell, NK-cell, and APC-mediated immune responses, etc.). The amino acid sequence in b) may have more than 90%, 93%, 95%, 97%, or 99% identity with SEQ ID NO. 1. The HLA-G1 fragment is typically of human origin.
In the construction method provided by the present invention, the first B2M fragment may include: c) A polypeptide fragment with an amino acid sequence shown as SEQ ID NO. 2; or, d) a polypeptide fragment having an amino acid sequence having more than 90% sequence identity to SEQ ID NO. 2 and having the function of the polypeptide fragment defined in c).
Amino acid sequence of endogenous B2M protein (first B2M fragment):
MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM(SEQ ID NO:2)
specifically, the amino acid sequence in d) specifically refers to: the amino acid sequence shown in SEQ ID NO. 2 is obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids at the N-terminal and/or C-terminal, and has the function of the polypeptide fragment shown in SEQ ID NO. 2. For example, the first B2M fragment typically has a beta sheet-like structure that primarily has the function of non-covalently binding to the major histocompatibility complex class I heavy chain. The amino acid sequence in d) may have more than 90%, 93%, 95%, 97%, or 99% identity with SEQ ID NO. 2. The first B2M fragment is typically of human origin.
In the construction method provided by the invention, the B2M-HLA-G1 fusion protein can further comprise a first flexible connecting peptide segment, wherein the first flexible connecting peptide segment is positioned between the HLA-G1 segment and the first B2M segment. The first flexible connecting peptide may generally be a flexible polypeptide of suitable length consisting of glycine (G), serine (S) and/or alanine (A) such that adjacent protein domains are free to move relative to each other, e.g., the amino acid sequence of the first flexible connecting peptide may comprise a polypeptide such as (GS) n 、(GGS) n 、(GGSG) n 、(GGGS) n A、(GGGGS) n A、(GGGGA) n A、(GGGGG) n A, etc., wherein n is selected from integers between 1 and 10. In some embodiments of the invention, the amino acid sequence of the first flexible connecting peptide may be 5-26 in length. In some embodiments of the invention, the first flexible connecting peptide segment may comprise a polypeptide segment having an amino acid sequence as shown in SEQ ID NO. 3. In other embodiments of the present invention, the B2M-HLA-G1 fusion protein comprises a first B2M fragment and an HLA-G1 fragment from the N-terminus to the C-terminus, and the B2M-HLA-G1 fusion protein comprises a polypeptide fragment with an amino acid sequence shown as SEQ ID NO. 4.
Flexible (G) 4 S) 4 Amino acid sequence of the connecting peptide fragment (first flexible connecting peptide fragment):
GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer(SEQ ID NO:3)
Amino acid sequence of B2M-HLA-G1 fusion protein:
a nucleic acid sequence encoding a B2M-HLA-G1 fusion protein, wherein endogenous B2M gene exon DNA sequences are bolded, intron DNA sequences are underlined:
in the construction method provided by the invention, the method for integrating exogenous nucleic acid encoding B2M-HLA-G1 fusion protein in the genome of the human pluripotent stem cell comprises the following steps: the gene encoding the HLA-G1 fragment was fused with the endogenous B2M gene in human pluripotent stem cells. In some embodiments of the invention, the gene encoding the HLA-G1 fragment may be substituted for the stop codon located in exon 3 of the endogenous B2M gene.
In the construction method provided by the present invention, the CIITA fragment may include: e) A polypeptide fragment with an amino acid sequence shown as SEQ ID NO. 5; or, f) a polypeptide fragment having an amino acid sequence having more than 90% sequence identity to SEQ ID NO. 5 and having the function of the polypeptide fragment defined in e).
Specifically, the amino acid sequence in f) specifically refers to: the amino acid sequence shown in SEQ ID NO. 5 is obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids at the N-terminal and/or C-terminal, and has the function of the polypeptide fragment shown in SEQ ID NO. 5. For example, the CIITA fragment typically has a dual or multifunctional domain that acts primarily as a transcriptional activator and plays a key role in the expression of Major Histocompatibility Complex (MHC) class II genes. The amino acid sequence in f) may have more than 90%, 93%, 95%, 97%, or 99% identity to SEQ ID NO. 5. The CIITA fragment is typically of human origin.
Amino acid sequence of CIITA protein:
MRCLAPRPAGSYLSEPQGSSQCATMELGPLEGGYLELLNSDADPLCLYHFYDQMDLAGEEEIELYSEPDTDTINCDQFSRLLCDMEGDEETREAYANIAELDQYVFQDSQLEGLSKDIFKHIGPDEVIGESMEMPAEVGQKSQKRPFPEELPADLKHWKPAEPPTVVTGSLLVRPVSDCSTLPCLPLPALFNQEPASGQMRLEKTDQIPMPFSSSSLSCLNLPEGPIQFVPTISTLPHGLWQISEAGTGVSSIFIYHGEVPQASQVPPPSGFTVHGLPTSPDRPGSTSPFAPSATDLPSMPEPALTSRANMTEHKTSPTQCPAAGEVSNKLPKWPEPVEQFYRSLQDTYGAEPAGPDGILVEVDLVQARLERSSSKSLERELATPDWAERQLAQGGLAEVLLAAKEHRRPRETRVIAVLGKAGQGKSYWAGAVSRAWACGRLPQYDFVFSVPCHCLNRPGDAYGLQDLLFSLGPQPLVAADEVFSHILKRPDRVLLILDGFEELEAQDGFLHSTCGPAPAEPCSLRGLLAGLFQKKLLRGCTLLLTARPRGRLVQSLSKADALFELSGFSMEQAQAYVMRYFESSGMTEHQDRALTLLRDRPLLLSHSHSPTLCRAVCQLSEALLELGEDAKLPSTLTGLYVGLLGRAALDSPPGALAELAKLAWELGRRHQSTLQEDQFPSADVRTWAMAKGLVQHPPRAAESELAFPSFLLQCFLGALWLALSGEIKDKELPQYLALTPRKKRPYDNWLEGVPRFLAGLIFQPPARCLGALLGPSAAASVDRKQKVLARYLKRLQPGTLRARQLLELLHCAHEAEEAGIWQHVVQELPGRLSFLGTRLTPPDAHVLGKALEAAGQDFSLDLRSTGICPSGLGSLVGLSCVTRFRAALSDTVALWESLQQHGETKLLQAAEEKFTIEPFKAKSLKDVEDLGKLVQTQRTRSSSEDTAGELPAVRDLKKLEFALGPVSGPQAFPKLVRILTAFSSLQHLDLDALSENKIGDEGVSQLSATFPQLKSLETLNLSQNNITDLGAYKLAEALPSLAASLLRLSLYNNCICDVGAESLARVLPDMVSLRVMDVQYNKFTAAGAQQLAASLRRCPHVETLAMWTPTIPFSVQEHLQQQDSRISLR(SEQ ID NO:5)
in the construction method provided by the invention, the method for not expressing CIITA specifically comprises the following steps: knocking out exon 3 of CIITA gene in human pluripotent stem cell genome; preferably, the knockout is performed by a gene editing method; more preferably, the gene editing is performed with gRNA (GGGAGGCTTATGCCAATAT) of the nucleotide sequence shown in SEQ ID NO. 11.
In the construction method provided by the present invention, the second B2M fragment may include: g) A polypeptide fragment with an amino acid sequence shown as SEQ ID NO. 2; or, h) a polypeptide fragment having an amino acid sequence which has more than 90% sequence identity to SEQ ID NO. 2 and which has the function of the polypeptide fragment defined in g).
Amino acid sequence of the second B2M fragment:
MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM(SEQ ID NO:2)
specifically, the amino acid sequence in h) specifically refers to: the amino acid sequence shown in SEQ ID NO. 2 is obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids at the N-terminal and/or C-terminal, and has the function of the polypeptide fragment shown in SEQ ID NO. 2. For example, the second B2M fragment typically has a beta sheet-like structure that primarily has the function of non-covalently binding to the major histocompatibility complex class I heavy chain. The amino acid sequence in h) may have more than 90%, 93%, 95%, 97%, or 99% identity to SEQ ID NO. 2. The second B2M fragment is typically of human origin.
In the construction method provided by the invention, the HLA-G5 fragment comprises the following components: i) A polypeptide fragment with an amino acid sequence shown as SEQ ID NO. 6; or, j) a polypeptide fragment having an amino acid sequence having more than 90% sequence identity to SEQ ID NO. 6 and having the function of the polypeptide fragment defined in i).
Amino acid sequence of HLA-G5 heavy chain sequence:
GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL*(SEQ ID NO:6)
specifically, the amino acid sequence in j) specifically refers to: the amino acid sequence shown in SEQ ID NO. 6 is obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids at the N-terminal and/or C-terminal, and has the function of the polypeptide fragment shown in SEQ ID NO. 6. For example, the HLA-G5 fragment typically has an intact extracellular structure of the alpha heavy chain, and unlike classical HLA I molecules, it has mainly immunosuppressive functions (e.g., can bind to inhibitory receptors, mainly including ILT2/CD85j/LILRB1, ILT4/CD85d/LILRB2, and KIR2DL4/CD158d, etc., and thus can modulate B-cell, T-cell, NK-cell, and APC-mediated immune responses, etc.). The amino acid sequence in j) may have more than 90%, 93%, 95%, 97%, or 99% identity with SEQ ID NO. 6 (Sequence identity). The HLA-G5 fragment is typically of human origin.
In the construction method provided by the invention, the B2M-HLA-G5 fusion protein can further comprise a second flexible connecting peptide segment, wherein the second flexible connecting peptide segment is positioned between the HLA-G5 segment and the second B2M segment. The second flexible linker peptide may typically be a flexible polypeptide of suitable length consisting of glycine (G), serine (S) and/or alanine (a) such that adjacent protein domains are free to move relative to each other, e.g. the amino acid sequence of the second flexible linker peptide may comprise sequences such as (GS) n, (GGS) n, (GGSG) n, (GGGS) nA, (GGGGS) nA, (GGGGA) nA, (ggggggg) nA, etc., wherein n is selected from integers between 1-10. In some embodiments of the invention, the amino acid sequence of the second flexible connecting peptide may be 5-26 in length. In some embodiments of the invention, the second flexible connecting peptide segment may comprise a polypeptide segment having an amino acid sequence as shown in SEQ ID NO. 5. In another embodiment of the invention, the B2M-HLA-G5 fusion protein comprises a second B2M fragment and an HLA-G5 fragment from the N end to the C end, and the B2M-HLA-G5 fusion protein comprises a polypeptide fragment with an amino acid sequence shown as SEQ ID NO. 8.
Amino acid sequence of flexible (G4S) 4 linker peptide (second flexible linker peptide):
GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer(SEQ ID NO:3)
amino acid sequence of B2M-HLA-G5 fusion protein:
nucleic acid sequence encoding B2M-HLA-G5 fusion protein:
ATGTCTCGCtccgtggccttagctgtgctcgcgctactctctctttctggcctggaggctatccagcg tactccaaagattcaggtttactcacgtcatccagcagagaatggaaagtcaaatttcctgaattgctatgtgtct gggtttcatccatccgacattgaagttgacttactgaagaatggagagagaattgaaaaagtggagcattcagact tgtctttcagcaaggactggtctttctatctcttgtactacactgaattcacccccactgaaaaagatgagtatgc ctgccgtgtgaaccatgtgactttgtcacagcccaagATAGTTAAGTGGGATCGAGACATGGGTGGAGGTGGAAGTGGTGGAGGTGGAAGTGGTGGAGGTGGAAGTGGTGGAGGTGGaagtGGCTCCCACTCCATGAGGTATttcagcgccgccgtgtcccggcccggccgcggggagccccgcttcatcgccatgggctacgtggacgacacgcagttcgtgcggttcgacagcgactcggcgtgtccgaggatggagccgcgggcgccgtgggtggagcaggaggggccggagtattgggaagaggagacacggaacaccaaggcccacgcacagactgacagaatgaacctgcagaccctgcgcggctactacaaccagagcgaggccagttctcacaccctccagtggatgattggctgcgacctggggtccgacggacgcctcctccgcgggtatgaacagtatgcctacgatggcaaggattacctcgccctgaacgaggacctgcgctcctggaccgcagcggacactgcggctcagatctccaagcgcaagtgtgaggcggccaatgtggctgaacaaaggagagcctacctggagggcacgtgcgtggagtggctccacagatacctggagaacgggaaggagatgctgcagcgcgcggacccccccaagacacacgtgacccaccaccctgtctttgactatgaggccaccctgaggtgctgggccctgggcttctaccctgcggagatcatactgacctggcagcgggatggggaggaccagacccaggacgtggagctcgtggagaccaggcctgcaggggatggaaccttccagaagtgggcagctgtggtggtgccttctggagaggagcagagatacacgtgccatgtgcagcatgaggggctgccggagcccctcatgctgagatggagtaaggagggagatggaggcatcatgtctgttagggaaagcaggagcctctctgaagacCTTTAA(SEQ ID NO:10)
in the construction method provided by the invention, the method for integrating exogenous nucleic acid encoding B2M-HLA-G5 fusion protein in the genome of the human pluripotent stem cell comprises the following steps: the nucleic acid encoding the B2M-HLA-G5 fusion protein is integrated into the genome of the human pluripotent stem cell by a lentiviral vector, so that the B2M-HLA-G5 fusion protein can be expressed.
The construction method of the human pluripotent stem cell provided by the invention can successfully integrate HLA-G1 fragment sequences at two endogenous B2M sites of the human pluripotent stem cell, knock out CIITA fragment sequences, and integrate sequences encoding B2M-HLA-G5 fusion proteins into the genome of the human pluripotent stem cell through a lentiviral vector. The constructed human pluripotent stem cell line endogenous HLA-A, -B, -C protein cannot reach the surface of a cell membrane. Meanwhile, CIITA is knocked out, so that pluripotent stem cells with excellent immune compatibility and unaffected multipotency and proliferation capacity are obtained; further, the pluripotent stem cells can be used as a source of cells of interest for cell transplantation.
In some embodiments, disrupting expression of a gene in a pluripotent stem cell is generally referred to herein as a gene "knockout". Typically, the technique to accomplish both failures is the same. Means by which gene knockout can be achieved include, but are not limited to ZFN, TALEN, and CRISPR/Cas9 technologies. Among particularly useful embodiments is disruption of the gene using CRISPR/Cas9 technology. In some cases, CRISPR/Cas9 technology is used to introduce small deletions/insertions into the coding region of a gene such that no functional protein is produced, e.g., frame shift mutations occur, which result in the production of stop codons such that truncated, non-functional proteins are produced.
In order to prevent CIITA or B2M from being expressed as a target gene, other techniques for down-regulating the expression of the target gene are also optional. Alternatively, such interfering RNA molecules may be prepared and obtained using interfering RNA molecules specific for the gene of interest (e.g., siRNA, shRNA, miRNA, etc.), based on CIITA or B2M sequence information provided in the present invention. The interfering RNA may be delivered into the cell by using an appropriate transfection reagent, or may also be delivered into the cell using a variety of techniques known in the art. siRNAs are typically about 21 nucleotides in length (e.g., 21-23 nucleotides). After introduction of the small RNA or RNAi into the cell, the sequence is passed to an enzyme complex called RISC (RNA-induced silencing complex), which recognizes the target and cleaves it with an endonuclease. As another alternative, the interference is performed using shRNA technology. shRNA is an RNA sequence that can be used to spin a compact hairpin and can be used to silence gene expression by RNA interference. shRNA uses a vector introduced into the cell and uses a promoter (e.g., U6) to ensure that shRNA is always expressed. As another alternative, antisense compounds that specifically hybridize to the gene of interest are utilized to modulate the expression of the gene of interest. Specific hybridization of an oligomer to its target nucleic acid interferes with the normal function of the nucleic acid. Such modulation of target nucleic acid function by a compound that specifically hybridizes to the target nucleic acid is commonly referred to as "antisense". Alternatively, homologous recombination methods may be used to specifically target the gene of interest for defective or absent expression. Nonetheless, CRISPR-based schemes are preferred embodiments. It will be appreciated by those skilled in the art that human pluripotent stem cells constructed by the method for constructing human pluripotent stem cells provided by the present invention may also be included in the present invention.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions such as those described in J.Sam Brookfield et al, molecular cloning guidelines, third edition, scientific Press, or according to the manufacturer's recommendations.
Unless stated otherwise, the following data statistics are performed by Graphpad software, and the comparison between two groups of data adopts a t-test method to test whether the data have statistical significance; comparison between 3-4 sets of data used one-way ANOVA (one-way ANOVA) with the Dunnett method for post hoc testing. P values less than 0.05 are considered statistically significant.
EXAMPLE 1 construction of B2M/CIITA double knockout hPSCs (DKO-hPSCs)
In a subsequent example of the invention, hPSCs were used from human embryonic stem cells, H9 strain.
To compare CIITA -/- Immunocompatibilities of B2Mm/sHLAG-hPSCs derived cells B2M/CIITA double knockout hPSCs were constructed as controls. The B2M and CIITA genes were knocked out simultaneously using gene editing methods (CRISPR/Cas 9 system in this example).
According to the information of B2M and CIITA gene transcripts, through analysis and research, the inventor optimizes sequences suitable for gene editing positions, and designs gRNA targeting the genomic positions based on the sequences, wherein the gRNA sequences are shown in table 1. And respectively constructing the gRNA on plasmids started by the U6 promoter to obtain gRNA expression plasmids gRNA-B2M and gRNA-CIITA. In the construction of DKO-hPSCs, gRNA-B2M plasmid and Cas9-T2A-GFP (Addgene # 44719) plasmid are firstly transferred into hPSCs, low-density single-cell plating is carried out after 4 days of culture, genomic DNA PCR and sequencing analysis are manually carried out after monoclonal growth, and clone with non-3 integer multiple base deletion or insertion is selected as B2M knockout hPSCs.
Based on this, CIITA knockout hPSCs were further constructed in the same manner as above, but the gRNA plasmid was replaced with a gRNA-CIITA plasmid, and the CIITA knockout monoclonal was further picked. Knocked-out hPSCs were constructed by the above method. Clones with non-3 fold base frameshift mutations were obtained as B2M/CIITA double knockouts hPSCs (DKO-hPSCs) (FIG. 2A).
TABLE 1 gRNA sequence targeting the B2M, CIITA site
Site(s) gRNA sequence
B2M AAGATTCAGGTTTACTCAC(SEQ ID NO:7)
CIITA GGGAGGCTTATGCCAATAT(SEQ ID NO:11)
Example 2, CIITA -/- Construction of B2Mm/sHLAG-hPSCs
Construction of CIITA -/- The B2Mm/sHLAG-hPSCs method is that, in Based on the cell constructed by the patent application CN113528448A, the gene editing method (CRISPR/Cas 9 is adopted in the present embodiment) is used to knock out the CIITA gene, specifically (fig. 1):
coding flexibility (G) was inserted in wild type hPSCs using CRISPR/Cas9 gene editing methods before the B2M gene coding frame stop codon 4 S) 4 HLA-G1 coding genes (recombinant expression B2M-HLA-G1 fusion protein, SEQ ID NO: 4) connected by connecting peptide segments, and the cell line obtained by construction only expresses an HLA-G1/B2M complex, and other HLA I family molecules cannot be normally expressed on cell membranes due to lack of B2M subunits. Then, B2M- (G) is introduced into the genome of the above-mentioned cell 4 S) 4 -coding box of HLA-G5: the B2M CDS sequence without stop codon (SEQ ID NO: 2), (Gly) 4 Ser) 4 The flexible linker peptide coding sequence and HLA-G5 heavy chain coding sequence were integrated into BamHI, mluI of puromycin resistant lentiviral vector pLVX-CAG-Puro, and the resulting recombinant lentiviral vector was introduced into cells to obtain B2Mm/sHLAG hPSCs (see corresponding description in CN 113528448A).
Further, CIITA gene was knocked out by gene editing method (CRISPR/Cas 9 system was used in this example), gRNA-CIITA plasmid (wherein gRNA sequence is as shown in Table 1) and Cas9-T2A-GFP plasmid were transfected by LONZA nucleofection, and single clone was selected for DNA PCR sequencing, clone with non-3 multiple base frame shift mutation was selected to obtain CIITA knocked out B2Mm/sHLAG hPSCs, and immunocompatible hPSCs were constructed and named CIITA -/- B2Mm/sHLAG-hPSCs (FIG. 2B). In the clones used in the subsequent examples, 5 bases were deleted after ATGGAAGGTGATGAAGAGAC and 1 base was inserted after ATGGAAGGTGATGAAGAGACCAGGG in the mRNA sequence of CIITA, resulting in a frame shift mutation after position 90 or 92 of the CIITA protein and premature termination of the CIITA protein at position 124 or 126.
The cells can be further differentiated into immune compatible functional cells such as myocardial cells, endothelial cells, neural cells, NK cells and the like by utilizing tissue specific cell lines.
Example 3 identification of pluripotency markers
To identify DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs self-renewing ability wild-type hPSCs (wt-hPSCs) were used as controls. Identification of wt-hPSCs, DKO-hPSCs and CIITA by flow cytometry and immunofluorescence -/- Expression of the pluripotency markers of B2 Mm/sHLAG-hPSCs.
Flow cytometry identification method:
cultured wt-hPSCs, DKO-hPSCs and CIITA-/-B2Mm/sHLAG-hPSCs were grown to 70% confluency, ackutase digested into single cells, spun down to collect cells, fixed, membrane broken and incubated with hPSCs multipotent cell markers OCT4 and SSEA4 antibodies, then stained with APC fluorophore coupled secondary antibodies, and analyzed by flow cytometry. The control group used isotype control antibody. The detection result is shown in FIG. 3.
Cell immunofluorescence identification method:
the cultured wt-hPSCs, DKO-hPSCs and CIITA-/-B2Mm/sHLAG-hPSCs clones are grown to proper sizes, the culture medium is sucked off, then the membranes are fixed, the membranes are broken and incubated with multipotent cell markers OCT4 and SOX2 antibodies, then secondary antibody staining coupled with Alexa 488 fluorophores is utilized, and image acquisition is carried out by using a laser confocal microscope. The detection results are shown in FIG. 4.
As shown in fig. 3, the expression positive rate of the pluripotency markers OCT4 and SSEA4 in the three cells is above 98%; as shown in fig. 4, the pluripotency markers OCT4 and SOX2 both have good nuclear localization characteristics.
The data prove that the obtained DKO-hPSCs and CIITA -/- The B2Mm/sHLAG-hPSCs and the wt-hPSCs have no difference in expression of the pluripotency markers, which indicates that the self-renewal capacity is good.
Example 4 identification of HLA molecule expression
To identify whether cells constructed using the above method express HLA molecules, the present inventors first identified using flow cytometry.
Flow cytometry identification method:
wt-hPSCs, DKO-hPSCs and CIITA -/- Adding interferon gamma (IFN-gamma) into B2Mm/sHLAG-hPSCs cell in a culture system with final concentration of 100ng/ml, culturing for 48 hr, digesting into single cell with Ackutase, and centrifuging to collect cell for detecting HL A molecule is expressed. Because of the large number of HLA I and HLA II family members, representative HLA-A, -B, -C (which is represented by HLA I family) and HLA-DR (which is represented by HLA II family) are adopted to represent the wt-hPSCs, DKO-hPSCs and CIITA -/- Expression of HLA I and HLA II molecules in B2 Mm/sHLAG-hPSCs. HLA-A/B/C antibodies are coupled with FITC fluorescent groups, and HLA-DR antibodies are coupled with PE fluorescent groups. Single cells were collected and incubated directly with the corresponding antibodies and HLA I and HLA II molecule expression was detected using flow cytometry. HLA-G1 identification also employed the flow cytometry method described above.
HLA-A, -B, -C is a classical HLA I family molecular member, comparing wt-hPSCs, DKO-hPSCs and CIITA -/- Expression of HLA-A, -B, -C in B2Mm/sHLAG-hPSCs, it was found that HLA-A, -B, -C was expressed only in wt-hPSCs, its expression was not in DKO-hPSCs and CIITA -/- Detected in B2Mm/sHLAG-hPSCs (FIG. 5A); whereas HLA-G1 molecules with immunosuppressive effects are only present in CIITA -/- B2Mm/sHLAG-hPSCs (FIG. 5B). hPSCs were treated with interferon gamma (IFN-. Gamma.) for 48 hours and HLA-DR expression was flow tested, indicating that the fluorescent signal values in wt were significantly higher than DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs cells (FIG. 5C).
Immunoblotting was used to identify secretory HLA-G5 molecule expression.
Immunoblotting identification method for secretory HLA-G5 expression:
wt-hPSCs, DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs cells were cultured in hPSCs medium to 50% confluency, and then replaced with DMEM/F12 basal medium for 24 hours, the medium supernatant was collected, cell debris was filtered off with a 0.25 μm filter, and the supernatant was concentrated using a 10kd ultrafiltration tube. Before immunoblotting, loading buffer (containing beta-mercaptoethanol) was added to a final concentration of 1x, and the protein was denatured in a water bath at 95℃for 10 minutes, and the protein was separated by 10% SDS-PAGE gel electrophoresis, and the detection antibody was 5A6G7.
Results demonstrate CIITA -/- B2Mm/sHLAG-hPSCs expressed HLA-G5, wt-hPSCs and DKO-hPSCs did not express HLA-G5 (FIG. 5D).
Example 5 Induction differentiation and identification of endothelial cells and cardiomyocytes
hPSCs can be obtained by in vitro induction methods to obtain cells of various tissue types, in this example, the inventors differentiated hPSCs into endothelial and cardiomyocytes.
1. Differentiation of endothelial cells
wt-hPSCs, DKO-hPSCs and CIITA -/- The B2Mm/sHLAG-hPSCs were treated with differentiation medium RPMI1640+0.2% BSA and 6. Mu.M CHIR99021 for 2 days when they were grown to 100%, and then with RPMI1640+0.2% BSA+50ng/ml bFGF for 1 day, and then with RPMI1640+0.2% BSA+50ng/ml VEGF+50ng/ml BMP4 for 2 days. Endothelial cells were purified on day 5 using Meitian and gentle CD144 (VE-Cadherin) MicroBeads beads. Purified cells were further cultured using ECM medium.
Differentiated cells (endothelial cells, day 6; cardiomyocytes, day 14) were taken, ackutase was digested into single cells, and the cell mass was removed by filtration through a 70 μm sieve. Endothelial cells are directly incubated with the cells using APC-conjugated CD144 antibodies for detection; the myocardial cells are fixed and broken, and the myocardial cell marker cTNT antibody is used as a primary antibody, and the APC coupled anti-mouse IgG antibody is used as a secondary antibody for detection.
The results showed that the purified endothelial cells had good endothelial morphology and an extremely high expression ratio of endothelial cell marker CD144 in endothelial cell differentiation (fig. 6A). Thus, immune-compatible endothelial cells are differentiated.
2. Differentiation of cardiomyocytes
wt-hPSCs, DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs were grown to 100% confluence and then treated with differentiation medium RPMI1640+0.2% BSA+6. Mu.M CHIR99021 for 2 days, then with RPMI1640+0.2% BSA+5. Mu.M IWR-1 for 2 days, and then with RPMI1640+0.2% BSA every 2 days. The onset of beating cardiomyocytes was initiated on day 7, and designated as wt-CMs, DKO-CMs and CIITA-/-B2Mm/sHLAG-hPSCs, respectively, depending on the type of starting hPSCs.
The method for identifying myocardial cells by using cytoimmunofluorescence was the same as the method for detecting hPSCs pluripotency markers described in example 3, except that the primary antibody was replaced with a myocardial cell marker cTNT antibody and a gap junction Connecin 43 antibody.
Among differentiated cardiomyocytes, three hPSCs derived cardiomyocytes had good myofilament structure by myogenin alpha-ACTININ staining, and good gap junction (Connecin-43) was formed between cells (FIG. 6B). Immunofluorescence identified the expression of sarcomere alpha-ACTININ and gap protein Connexin-43 in differentiated cardiomyocytes as shown in figure 6C.
Thus, immune-compatible Cardiomyocytes (CIITA) -/- B2Mm/sHLAG-CMs)。
Example 6 immune recognition and killing action
To explore CIITA -/- B2Mm/sHLAG-hPSCs and whether derived cells thereof have the effect of recognizing and killing escape immune cells, the inventor adopts in vitro immune cells (PBMCs or NK 92) and CIITA -/- B2 Mm/sHLAG-hPSCs-derived cardiomyocytes were identified by co-culture. T cells are subjected to activation marker detection, proliferation detection, killing effect detection on target cells and secreted IFN-gamma detection. NK cells perform recognition and killing detection of target cells and detection of secreted IFN-gamma.
1. T cell activation marker detection
T cells are activated by mismatched HLA molecules, thus utilizing wt-CMs as controls and CIITA -/- B2Mm/sHLAG-hPSCs comparison of activation of T cells. wt-CMs and CIITA were combined -/- B2Mm/sHLAG-hPSCs were plated in 96-well plates and added to final concentration of 100ng/ml IFN-gamma for 48 hours to stimulate HLA molecule expression. Thereafter, IFN-. Gamma.was removed, primary peripheral blood mononuclear cells (PBMCs, which are enriched in T cells) were added at a ratio of myocardial cells to human peripheral blood mononuclear cells (PMBCs) of 1:3, and after 48 hours of co-culture, the co-cultured PBMCs were collected, and the T cell marker CD3 antibody and the T cell early activation marker CD69 antibody were incubated, followed by flow cytometry. Comparison of wt-CMs and CIITA by the proportion of CD69 positive cells in CD3 positive cells -/- B2Mm/sHLAG-hPSCs ability to activate T cells.
2. T cell proliferation assay
Pretreatment and "T cell activation marker detection", PBMCs were labeled with C prior to co-cultivationFSE fluorescent labeling followed by co-culture with cardiomyocytes for 7 days, followed by collection of PBMCs, incubation of T cell marker CD3 antibodies, and flow cytometry. CFSE does not leak from cells in the labeled cells, but dilutes with cell division, and thus CFSE fluorescence signal in proliferating T cells decreases, comparing wt-CMs to CIITA by comparing the proportion of cells with lower CFSE signal to all CD3 positive cells -/- B2Mm/sHLAG-hPSCs effect on T cell proliferation.
3. Detection of killing of target cells by T cells
Pre-treatment is associated with "T cell activation marker detection", PBMCs with wt-CMs or CIITA -/- B2Mm/sHLAG-hPSCs are co-cultured for 3-4 days, then a co-culture supernatant culture solution is collected, 300g is centrifuged for 3 minutes to remove cells and cell fragments, and then the lactic dehydrogenase activity in the supernatant is detected by using a Biyun-Tian LDH detection kit to determine the degree of cell damage.
4. IFN-gamma detection of PBMC secretion
Pre-treatment is associated with "T cell activation marker detection", PBMCs with wt-CMs or CIITA -/- B2Mm/sHLAG-hPSCs were co-cultured for 3-4 days, then the co-culture supernatant medium was collected, centrifuged at 300g for 3 minutes to remove cells and cell debris, and then IFN-gamma content in the supernatant was detected using IFN-gamma detection kit.
5. NK cell recognition and killing detection for target cells
NK cells recognize cells without HLA molecule expression, and therefore DKO-CMs were used as controls in this example. DKO-CMs and CIITA -/- B2Mm/sHLAG-hPSCs were plated in 96-well plates at 24 hours according to CMs: NK-92=10:1, 3:1 and 1:1 were added to NK-92 cells, the culture was performed for 48 hours, the culture broth of the co-culture supernatant was collected, and the cells and cell debris were removed by centrifugation at 300g for 3 minutes, and then the lactate dehydrogenase activity in the supernatant was detected by using the Biyun-Tian LDH detection kit to determine the degree of cell damage.
6. IFN-gamma detection of NK cell secretion
Cell pretreatment and 'NK cell recognition and killing detection' are carried out, co-culture is carried out for 3-4 days, co-culture supernatant is collected, 300g is centrifuged for 3 minutes to remove cells and cell fragments, and then IFN-gamma detection kit is used for detecting IFN-gamma content in the supernatant.
Human PBMCs were co-cultured with cardiomyocytes and early T cell activation marker CD69 expression was identified. The results showed that, compared to wt cardiomyocytes (wt-CMs), immunocompatible cardiomyocytes (CIITA -/- B2 Mm/hlag-CMs) significantly decreased activation of T cells; and CIITA -/- The effect of B2Mm/sHLAG-CMs on T cell proliferation was also significantly lower than that of wt-CMs. At the same time, PBMCs pair CIITA -/- The killing effect of B2Mm/sHLAG-CMs is significantly lower than that of wt-CMs, and CIITA -/- The ability of B2Mm/sHLAG-CMs to stimulate IFN-gamma secretion by PBMCs is also significantly lower than that of wt-CMs, as shown in FIGS. 7A-B.
To check CIITA -/- Activation of NK cells by B2Mm/sHLAG-CMs the inventors used DKO-CMs as a control to co-culture differentiated cardiomyocytes with NK-92 cells to find NK-92 vs CIITA -/- The killing effect of B2Mm/sHLAG-CMs cells is obviously lower than DKO-CMs, and CIITA -/- The ability of B2Mm/sHLAG-CMs to stimulate NK-92 secretion of IFN-gamma is also significantly lower than DKO-CMs, as shown in FIGS. 7C-F.
The above data demonstrate that CIITA -/- B2Mm/sHLAG-hPSCs derived cardiomyocytes have the recognition and killing function of escaping T cells and NK cells.
Example 7 identification of cell residence after transplantation of hPSCs after myocardial infarction
To further explore CIITA in a body injury environment -/- Whether B2Mm/sHLAG-hPSCs have better residence effect or not, the inventor utilizes an immune system humanized mouse (Hu-mice) to construct a heart injury model (an ischemia/reperfusion injury model is adopted in the embodiment), and myocardial cells from different hPSCs are transplanted in the modeled myocardium. The method comprises the following specific steps: at week 10 of immune reconstruction, human immune cells already occupy a considerable proportion of Hu-mice heart coronary left anterior descending branch in the peripheral blood of mice, and after 60 minutes of ischemia, the heart muscle ischemia/reperfusion (I/R) model is constructed. The model mice were randomly divided into I/R groups, I/R+wt-CMs groups, I/R+DKO-CMs groups, and I/R+CIITA groups -/- B2Mm/sHLAG-CMs group; cell transplantation was performed during reperfusion, and myocardial injection was performed in the border area of myocardial infarction for 5×10 per mouse 5 Amounts of wt-CMs, DKO-CMs and CIITA -/- B2Mm/sHLAG-CMs。
Mice hearts were sampled on day 28 post-transplantation and engrafted cells were identified by immunofluorescence and qRT-PCR. The method for detecting the residence of the transplanted cells comprises the following steps:
the mouse hearts were harvested 28 days after cell transplantation and OCT-embedded. The heart of the mouse is divided into 12 layers from the apex to the ligation point according to a certain interval, each layer of cells is frozen and sectioned, then the transplanted cells are identified by immunofluorescence, and the proportion of the transplanted cell area to the scar area is counted by Masson staining.
Immunofluorescence: tissues were fixed, permeabilized and blocked after frozen sections, and then the sarcomere alpha-ACTININ antibody was co-incubated with human KU80 antibody (C48E 7, CST). Images were collected using a fluorescence microscope and counted.
qRT-PCR: collecting Hu-mice heart samples 28 days after cell transplantation, extracting RNA, turning into cDNA, and identifying cell residence by using a human cTNT primer through fluorescent quantitative PCR.
By comparing cell residence area, CIITA was found in female and male mice -/- The residence area of B2Mm/sHLAG-CMs is significantly higher than that of wt-CMs and DKO-CMs. qRT-PCR results were consistent with immunofluorescent staining results (FIGS. 8A-D).
The above data demonstrate that CIITA -/- The long-term residence capacity of B2Mm/sHLAG-CMs in the injury model is significantly stronger than that of wt-CMs and DKO-CMs.
Example 8 repair of myocardial infarction area by transplanted cells
Further, the present inventors investigated the repair of damaged areas of the mouse heart by engrafting cell residency using Masson staining.
Masson staining method: after frozen sections, the tissues are fixed, then the tissues are stained by using a Biyun Masson staining kit, the re-muscle area and the scar area are counted, and the re-muscle ratio is calculated.
Re-muscular ratio = re-muscular area/scar area
The results show that cells at the damaged part gradually die after the heart injury of the mice, and scar tissues are formed.Comparison of cardiac sections of mice after three cell transplants shows CIITA -/- After B2Mm/sHLAG-CMs transplantation, myocardial tissue can be formed at the damaged part to compensate for the loss of myocardial cells.
Statistical results show CIITA -/- The extent of re-muscle formation in the damaged areas after B2Mm/sHLAG-CMs transplantation was significantly higher than that of wt-CMs and DKO-CMs (FIGS. 9A-B).
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims. All documents referred to in this application are incorporated by reference herein as if each was individually incorporated by reference.
Sequence listing
<110> Shanghai nutrition and health institute of China academy of sciences
Shanghai Oriental Hospital (Oriental Hospital Affiliated to Tongji University)
Tongji University
<120> immune compatible human pluripotent stem cell, preparation method and application thereof
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<400> 1
Gly Ser His Ser Met Arg Tyr Phe Ser Ala Ala Val Ser Arg Pro Gly
1 5 10 15
Arg Gly Glu Pro Arg Phe Ile Ala Met Gly Tyr Val Asp Asp Thr Gln
20 25 30
Phe Val Arg Phe Asp Ser Asp Ser Ala Cys Pro Arg Met Glu Pro Arg
35 40 45
Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Glu Glu Glu Thr
50 55 60
Arg Asn Thr Lys Ala His Ala Gln Thr Asp Arg Met Asn Leu Gln Thr
65 70 75 80
Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala Ser Ser His Thr Leu Gln
85 90 95
Trp Met Ile Gly Cys Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg Gly
100 105 110
Tyr Glu Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Leu Ala Leu Asn Glu
115 120 125
Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Ser Lys
130 135 140
Arg Lys Cys Glu Ala Ala Asn Val Ala Glu Gln Arg Arg Ala Tyr Leu
145 150 155 160
Glu Gly Thr Cys Val Glu Trp Leu His Arg Tyr Leu Glu Asn Gly Lys
165 170 175
Glu Met Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His
180 185 190
Pro Val Phe Asp Tyr Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe
195 200 205
Tyr Pro Ala Glu Ile Ile Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln
210 215 220
Thr Gln Asp Val Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr
225 230 235 240
Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg
245 250 255
Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Met Leu
260 265 270
Arg Trp Lys Gln Ser Ser Leu Pro Thr Ile Pro Ile Met Gly Ile Val
275 280 285
Ala Gly Leu Val Val Leu Ala Ala Val Val Thr Gly Ala Ala Val Ala
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Ala Val Leu Trp Arg Lys Lys Ser Ser Asp
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Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser
1 5 10 15
Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg
20 25 30
His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser
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Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu
50 55 60
Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp
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Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp
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Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile
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Val Lys Trp Asp Arg Asp Met
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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
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Gly Gly Gly Ser
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<221> PEPTIDE
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Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser
1 5 10 15
Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg
20 25 30
His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser
35 40 45
Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu
50 55 60
Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp
65 70 75 80
Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp
85 90 95
Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile
100 105 110
Val Lys Trp Asp Arg Asp Met Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser His Ser Met
130 135 140
Arg Tyr Phe Ser Ala Ala Val Ser Arg Pro Gly Arg Gly Glu Pro Arg
145 150 155 160
Phe Ile Ala Met Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp
165 170 175
Ser Asp Ser Ala Cys Pro Arg Met Glu Pro Arg Ala Pro Trp Val Glu
180 185 190
Gln Glu Gly Pro Glu Tyr Trp Glu Glu Glu Thr Arg Asn Thr Lys Ala
195 200 205
His Ala Gln Thr Asp Arg Met Asn Leu Gln Thr Leu Arg Gly Tyr Tyr
210 215 220
Asn Gln Ser Glu Ala Ser Ser His Thr Leu Gln Trp Met Ile Gly Cys
225 230 235 240
Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg Gly Tyr Glu Gln Tyr Ala
245 250 255
Tyr Asp Gly Lys Asp Tyr Leu Ala Leu Asn Glu Asp Leu Arg Ser Trp
260 265 270
Thr Ala Ala Asp Thr Ala Ala Gln Ile Ser Lys Arg Lys Cys Glu Ala
275 280 285
Ala Asn Val Ala Glu Gln Arg Arg Ala Tyr Leu Glu Gly Thr Cys Val
290 295 300
Glu Trp Leu His Arg Tyr Leu Glu Asn Gly Lys Glu Met Leu Gln Arg
305 310 315 320
Ala Asp Pro Pro Lys Thr His Val Thr His His Pro Val Phe Asp Tyr
325 330 335
Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile
340 345 350
Ile Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Val Glu
355 360 365
Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala
370 375 380
Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr Cys His Val
385 390 395 400
Gln His Glu Gly Leu Pro Glu Pro Leu Met Leu Arg Trp Lys Gln Ser
405 410 415
Ser Leu Pro Thr Ile Pro Ile Met Gly Ile Val Ala Gly Leu Val Val
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Leu Ala Ala Val Val Thr Gly Ala Ala Val Ala Ala Val Leu Trp Arg
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Lys Lys Ser Ser Asp
450
<210> 5
<211> 1130
<212> PRT
<213> Artificial Sequence
<220>
<221> PEPTIDE
<222> (1)..(1130)
<223> amino acid sequence of CIITA protein
<400> 5
Met Arg Cys Leu Ala Pro Arg Pro Ala Gly Ser Tyr Leu Ser Glu Pro
1 5 10 15
Gln Gly Ser Ser Gln Cys Ala Thr Met Glu Leu Gly Pro Leu Glu Gly
20 25 30
Gly Tyr Leu Glu Leu Leu Asn Ser Asp Ala Asp Pro Leu Cys Leu Tyr
35 40 45
His Phe Tyr Asp Gln Met Asp Leu Ala Gly Glu Glu Glu Ile Glu Leu
50 55 60
Tyr Ser Glu Pro Asp Thr Asp Thr Ile Asn Cys Asp Gln Phe Ser Arg
65 70 75 80
Leu Leu Cys Asp Met Glu Gly Asp Glu Glu Thr Arg Glu Ala Tyr Ala
85 90 95
Asn Ile Ala Glu Leu Asp Gln Tyr Val Phe Gln Asp Ser Gln Leu Glu
100 105 110
Gly Leu Ser Lys Asp Ile Phe Lys His Ile Gly Pro Asp Glu Val Ile
115 120 125
Gly Glu Ser Met Glu Met Pro Ala Glu Val Gly Gln Lys Ser Gln Lys
130 135 140
Arg Pro Phe Pro Glu Glu Leu Pro Ala Asp Leu Lys His Trp Lys Pro
145 150 155 160
Ala Glu Pro Pro Thr Val Val Thr Gly Ser Leu Leu Val Arg Pro Val
165 170 175
Ser Asp Cys Ser Thr Leu Pro Cys Leu Pro Leu Pro Ala Leu Phe Asn
180 185 190
Gln Glu Pro Ala Ser Gly Gln Met Arg Leu Glu Lys Thr Asp Gln Ile
195 200 205
Pro Met Pro Phe Ser Ser Ser Ser Leu Ser Cys Leu Asn Leu Pro Glu
210 215 220
Gly Pro Ile Gln Phe Val Pro Thr Ile Ser Thr Leu Pro His Gly Leu
225 230 235 240
Trp Gln Ile Ser Glu Ala Gly Thr Gly Val Ser Ser Ile Phe Ile Tyr
245 250 255
His Gly Glu Val Pro Gln Ala Ser Gln Val Pro Pro Pro Ser Gly Phe
260 265 270
Thr Val His Gly Leu Pro Thr Ser Pro Asp Arg Pro Gly Ser Thr Ser
275 280 285
Pro Phe Ala Pro Ser Ala Thr Asp Leu Pro Ser Met Pro Glu Pro Ala
290 295 300
Leu Thr Ser Arg Ala Asn Met Thr Glu His Lys Thr Ser Pro Thr Gln
305 310 315 320
Cys Pro Ala Ala Gly Glu Val Ser Asn Lys Leu Pro Lys Trp Pro Glu
325 330 335
Pro Val Glu Gln Phe Tyr Arg Ser Leu Gln Asp Thr Tyr Gly Ala Glu
340 345 350
Pro Ala Gly Pro Asp Gly Ile Leu Val Glu Val Asp Leu Val Gln Ala
355 360 365
Arg Leu Glu Arg Ser Ser Ser Lys Ser Leu Glu Arg Glu Leu Ala Thr
370 375 380
Pro Asp Trp Ala Glu Arg Gln Leu Ala Gln Gly Gly Leu Ala Glu Val
385 390 395 400
Leu Leu Ala Ala Lys Glu His Arg Arg Pro Arg Glu Thr Arg Val Ile
405 410 415
Ala Val Leu Gly Lys Ala Gly Gln Gly Lys Ser Tyr Trp Ala Gly Ala
420 425 430
Val Ser Arg Ala Trp Ala Cys Gly Arg Leu Pro Gln Tyr Asp Phe Val
435 440 445
Phe Ser Val Pro Cys His Cys Leu Asn Arg Pro Gly Asp Ala Tyr Gly
450 455 460
Leu Gln Asp Leu Leu Phe Ser Leu Gly Pro Gln Pro Leu Val Ala Ala
465 470 475 480
Asp Glu Val Phe Ser His Ile Leu Lys Arg Pro Asp Arg Val Leu Leu
485 490 495
Ile Leu Asp Gly Phe Glu Glu Leu Glu Ala Gln Asp Gly Phe Leu His
500 505 510
Ser Thr Cys Gly Pro Ala Pro Ala Glu Pro Cys Ser Leu Arg Gly Leu
515 520 525
Leu Ala Gly Leu Phe Gln Lys Lys Leu Leu Arg Gly Cys Thr Leu Leu
530 535 540
Leu Thr Ala Arg Pro Arg Gly Arg Leu Val Gln Ser Leu Ser Lys Ala
545 550 555 560
Asp Ala Leu Phe Glu Leu Ser Gly Phe Ser Met Glu Gln Ala Gln Ala
565 570 575
Tyr Val Met Arg Tyr Phe Glu Ser Ser Gly Met Thr Glu His Gln Asp
580 585 590
Arg Ala Leu Thr Leu Leu Arg Asp Arg Pro Leu Leu Leu Ser His Ser
595 600 605
His Ser Pro Thr Leu Cys Arg Ala Val Cys Gln Leu Ser Glu Ala Leu
610 615 620
Leu Glu Leu Gly Glu Asp Ala Lys Leu Pro Ser Thr Leu Thr Gly Leu
625 630 635 640
Tyr Val Gly Leu Leu Gly Arg Ala Ala Leu Asp Ser Pro Pro Gly Ala
645 650 655
Leu Ala Glu Leu Ala Lys Leu Ala Trp Glu Leu Gly Arg Arg His Gln
660 665 670
Ser Thr Leu Gln Glu Asp Gln Phe Pro Ser Ala Asp Val Arg Thr Trp
675 680 685
Ala Met Ala Lys Gly Leu Val Gln His Pro Pro Arg Ala Ala Glu Ser
690 695 700
Glu Leu Ala Phe Pro Ser Phe Leu Leu Gln Cys Phe Leu Gly Ala Leu
705 710 715 720
Trp Leu Ala Leu Ser Gly Glu Ile Lys Asp Lys Glu Leu Pro Gln Tyr
725 730 735
Leu Ala Leu Thr Pro Arg Lys Lys Arg Pro Tyr Asp Asn Trp Leu Glu
740 745 750
Gly Val Pro Arg Phe Leu Ala Gly Leu Ile Phe Gln Pro Pro Ala Arg
755 760 765
Cys Leu Gly Ala Leu Leu Gly Pro Ser Ala Ala Ala Ser Val Asp Arg
770 775 780
Lys Gln Lys Val Leu Ala Arg Tyr Leu Lys Arg Leu Gln Pro Gly Thr
785 790 795 800
Leu Arg Ala Arg Gln Leu Leu Glu Leu Leu His Cys Ala His Glu Ala
805 810 815
Glu Glu Ala Gly Ile Trp Gln His Val Val Gln Glu Leu Pro Gly Arg
820 825 830
Leu Ser Phe Leu Gly Thr Arg Leu Thr Pro Pro Asp Ala His Val Leu
835 840 845
Gly Lys Ala Leu Glu Ala Ala Gly Gln Asp Phe Ser Leu Asp Leu Arg
850 855 860
Ser Thr Gly Ile Cys Pro Ser Gly Leu Gly Ser Leu Val Gly Leu Ser
865 870 875 880
Cys Val Thr Arg Phe Arg Ala Ala Leu Ser Asp Thr Val Ala Leu Trp
885 890 895
Glu Ser Leu Gln Gln His Gly Glu Thr Lys Leu Leu Gln Ala Ala Glu
900 905 910
Glu Lys Phe Thr Ile Glu Pro Phe Lys Ala Lys Ser Leu Lys Asp Val
915 920 925
Glu Asp Leu Gly Lys Leu Val Gln Thr Gln Arg Thr Arg Ser Ser Ser
930 935 940
Glu Asp Thr Ala Gly Glu Leu Pro Ala Val Arg Asp Leu Lys Lys Leu
945 950 955 960
Glu Phe Ala Leu Gly Pro Val Ser Gly Pro Gln Ala Phe Pro Lys Leu
965 970 975
Val Arg Ile Leu Thr Ala Phe Ser Ser Leu Gln His Leu Asp Leu Asp
980 985 990
Ala Leu Ser Glu Asn Lys Ile Gly Asp Glu Gly Val Ser Gln Leu Ser
995 1000 1005
Ala Thr Phe Pro Gln Leu Lys Ser Leu Glu Thr Leu Asn Leu Ser Gln
1010 1015 1020
Asn Asn Ile Thr Asp Leu Gly Ala Tyr Lys Leu Ala Glu Ala Leu Pro
1025 1030 1035 1040
Ser Leu Ala Ala Ser Leu Leu Arg Leu Ser Leu Tyr Asn Asn Cys Ile
1045 1050 1055
Cys Asp Val Gly Ala Glu Ser Leu Ala Arg Val Leu Pro Asp Met Val
1060 1065 1070
Ser Leu Arg Val Met Asp Val Gln Tyr Asn Lys Phe Thr Ala Ala Gly
1075 1080 1085
Ala Gln Gln Leu Ala Ala Ser Leu Arg Arg Cys Pro His Val Glu Thr
1090 1095 1100
Leu Ala Met Trp Thr Pro Thr Ile Pro Phe Ser Val Gln Glu His Leu
1105 1110 1115 1120
Gln Gln Gln Asp Ser Arg Ile Ser Leu Arg
1125 1130
<210> 6
<211> 295
<212> PRT
<213> Artificial Sequence
<220>
<221> CHAIN
<222> (1)..(295)
<223> amino acid sequence of HLA-G5 heavy chain sequence
<400> 6
Gly Ser His Ser Met Arg Tyr Phe Ser Ala Ala Val Ser Arg Pro Gly
1 5 10 15
Arg Gly Glu Pro Arg Phe Ile Ala Met Gly Tyr Val Asp Asp Thr Gln
20 25 30
Phe Val Arg Phe Asp Ser Asp Ser Ala Cys Pro Arg Met Glu Pro Arg
35 40 45
Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Glu Glu Glu Thr
50 55 60
Arg Asn Thr Lys Ala His Ala Gln Thr Asp Arg Met Asn Leu Gln Thr
65 70 75 80
Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala Ser Ser His Thr Leu Gln
85 90 95
Trp Met Ile Gly Cys Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg Gly
100 105 110
Tyr Glu Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Leu Ala Leu Asn Glu
115 120 125
Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Ser Lys
130 135 140
Arg Lys Cys Glu Ala Ala Asn Val Ala Glu Gln Arg Arg Ala Tyr Leu
145 150 155 160
Glu Gly Thr Cys Val Glu Trp Leu His Arg Tyr Leu Glu Asn Gly Lys
165 170 175
Glu Met Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His
180 185 190
Pro Val Phe Asp Tyr Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe
195 200 205
Tyr Pro Ala Glu Ile Ile Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln
210 215 220
Thr Gln Asp Val Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr
225 230 235 240
Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg
245 250 255
Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Met Leu
260 265 270
Arg Trp Ser Lys Glu Gly Asp Gly Gly Ile Met Ser Val Arg Glu Ser
275 280 285
Arg Ser Leu Ser Glu Asp Leu
290 295
<210> 7
<211> 19
<212> DNA/RNA
<213> Artificial Sequence
<220>
<221> misc_RNA
<222> (1)..(19)
<223> gRNA sequence of B2M site
<400> 7
aagattcagg tttactcac 19
<210> 8
<211> 434
<212> PRT
<213> Artificial Sequence
<220>
<221> CHAIN
<222> (1)..(434)
<223> amino acid sequence of B2M-HLA-G5 fusion protein
<400> 8
Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser
1 5 10 15
Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg
20 25 30
His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser
35 40 45
Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu
50 55 60
Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp
65 70 75 80
Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp
85 90 95
Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile
100 105 110
Val Lys Trp Asp Arg Asp Met Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser His Ser Met
130 135 140
Arg Tyr Phe Ser Ala Ala Val Ser Arg Pro Gly Arg Gly Glu Pro Arg
145 150 155 160
Phe Ile Ala Met Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp
165 170 175
Ser Asp Ser Ala Cys Pro Arg Met Glu Pro Arg Ala Pro Trp Val Glu
180 185 190
Gln Glu Gly Pro Glu Tyr Trp Glu Glu Glu Thr Arg Asn Thr Lys Ala
195 200 205
His Ala Gln Thr Asp Arg Met Asn Leu Gln Thr Leu Arg Gly Tyr Tyr
210 215 220
Asn Gln Ser Glu Ala Ser Ser His Thr Leu Gln Trp Met Ile Gly Cys
225 230 235 240
Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg Gly Tyr Glu Gln Tyr Ala
245 250 255
Tyr Asp Gly Lys Asp Tyr Leu Ala Leu Asn Glu Asp Leu Arg Ser Trp
260 265 270
Thr Ala Ala Asp Thr Ala Ala Gln Ile Ser Lys Arg Lys Cys Glu Ala
275 280 285
Ala Asn Val Ala Glu Gln Arg Arg Ala Tyr Leu Glu Gly Thr Cys Val
290 295 300
Glu Trp Leu His Arg Tyr Leu Glu Asn Gly Lys Glu Met Leu Gln Arg
305 310 315 320
Ala Asp Pro Pro Lys Thr His Val Thr His His Pro Val Phe Asp Tyr
325 330 335
Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile
340 345 350
Ile Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Val Glu
355 360 365
Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala
370 375 380
Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr Cys His Val
385 390 395 400
Gln His Glu Gly Leu Pro Glu Pro Leu Met Leu Arg Trp Ser Lys Glu
405 410 415
Gly Asp Gly Gly Ile Met Ser Val Arg Glu Ser Arg Ser Leu Ser Glu
420 425 430
Asp Leu
<210> 9
<211> 5798
<212> DNA/RNA
<213> Artificial Sequence
<220>
<221> gene
<222> (1)..(5798)
<223> nucleic acid sequence encoding B2M-HLA-G1 fusion protein
<400> 9
atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctctttctgg cctggaggct 60
atccagcgtg agtctctcct accctcccgc tctggtcctt cctctcccgc tctgcaccct 120
ctgtggccct cgctgtgctc tctcgctccg tgacttccct tctccaagtt ctccttggtg 180
gcccgccgtg gggctagtcc agggctggat ctcggggaag cggcggggtg gcctgggagt 240
ggggaagggg gtgcgcaccc gggacgcgcg ctacttgccc ctttcggcgg ggagcagggg 300
agacctttgg cctacggcga cgggagggtc gggacaaagt ttagggcgtc gataagcgtc 360
agagcgccga ggttggggga gggtttctct tccgctcttt cgcggggcct ctggctcccc 420
cagcgcagct ggagtggggg acgggtaggc tcgtcccaaa ggcgcggcgc tgaggtttgt 480
gaacgcgtgg aggggcgctt ggggtctggg ggaggcgtcg cccgggtaag cctgtctgct 540
gcggctctgc ttcccttaga ctggagagct gtggacttcg tctaggcgcc cgctaagttc 600
gcatgtccta gcacctctgg gtctatgtgg ggccacaccg tggggaggaa acagcacgcg 660
acgtttgtag aatgcttggc tgtgatacaa agcggtttcg aataattaac ttatttgttc 720
ccatcacatg tcacttttaa aaaattataa gaactacccg ttattgacat ctttctgtgt 780
gccaaggact ttatgtgctt tgcgtcattt aattttgaaa acagttatct tccgccatag 840
ataactacta tggttatctt ctgcctctca cagatgaaga aactaaggca ccgagatttt 900
aagaaactta attacacagg ggataaatgg cagcaatcga gattgaagtc aagcctaacc 960
agggcttttg cgggagcgca tgccttttgg ctgtaattcg tgcatttttt tttaagaaaa 1020
acgcctgcct tctgcgtgag attctccaga gcaaactggg cggcatgggc cctgtggtct 1080
tttcgtacag agggcttcct ctttggctct ttgcctggtt gtttccaaga tgtactgtgc 1140
ctcttacttt cggttttgaa aacatgaggg ggttgggcgt ggtagcttac gcctgtaatc 1200
ccagcactta gggaggccga ggcgggagga tggcttgagg tccgtagttg agaccagcct 1260
ggccaacatg gtgaagcctg gtctctacaa aaaataataa caaaaattag ccgggtgtgg 1320
tggctcgtgc ctgtggtccc agctgctccg gtggctgagg cgggaggatc tcttgagctt 1380
aggcttttga gctatcatgg cgccagtgca ctccagcgtg ggcaacagag cgagaccctg 1440
tctctcaaaa aagaaaaaaa aaaaaaaaga aagagaaaag aaaagaaaga aagaagtgaa 1500
ggtttgtcag tcaggggagc tgtaaaacca ttaataaaga taatccaaga tggttaccaa 1560
gactgttgag gacgccagag atcttgagca ctttctaagt acctggcaat acactaagcg 1620
cgctcacctt ttcctctggc aaaacatgat cgaaagcaga atgttttgat catgagaaaa 1680
ttgcatttaa tttgaataca atttatttac aacataaagg ataatgtata tatcaccacc 1740
attactggta tttgctggtt atgttagatg tcattttaaa aaataacaat ctgatattta 1800
aaaaaaaatc ttattttgaa aatttccaaa gtaatacatg ccatgcatag accatttctg 1860
gaagatacca caagaaacat gtaatgatga ttgcctctga aggtctattt tcctcctctg 1920
acctgtgtgt gggttttgtt tttgttttac tgtgggcata aattaatttt tcagttaagt 1980
tttggaagct taaataactc tccaaaagtc ataaagccag taactggttg agcccaaatt 2040
caaacccagc ctgtctgata cttgtcctct tcttagaaaa gattacagtg atgctctcac 2100
aaaatcttgc cgccttccct caaacagaga gttccaggca ggatgaatct gtgctctgat 2160
ccctgaggca tttaatatgt tcttattatt agaagctcag atgcaaagag ctctcttagc 2220
ttttaatgtt atgaaaaaaa tcaggtcttc attagattcc ccaatccacc tcttgatggg 2280
gctagtagcc tttccttaat gatagggtgt ttctagagag atatatctgg tcaaggtggc 2340
ctggtactcc tccttctccc cacagcctcc cagacaagga ggagtagctg ccttttagtg 2400
atcatgtacc ctgaatataa gtgtatttaa aagaatttta tacacatata tttagtgtca 2460
atctgtatat ttagtagcac taacacttct cttcattttc aatgaaaaat atagagttta 2520
taatattttc ttcccacttc cccatggatg gtctagtcat gcctctcatt ttggaaagta 2580
ctgtttctga aacattaggc aatatattcc caacctggct agtttacagc aatcacctgt 2640
ggatgctaat taaaacgcaa atcccactgt cacatgcatt actccatttg atcataatgg 2700
aaagtatgtt ctgtcccatt tgccatagtc ctcacctatc cctgttgtat tttatcgggt 2760
ccaactcaac catttaaggt atttgccagc tcttgtatgc atttaggttt tgtttctttg 2820
ttttttagct catgaaatta ggtacaaagt cagagagggg tctggcatat aaaacctcag 2880
cagaaataaa gaggttttgt tgtttggtaa gaacatacct tgggttggtt gggcacggtg 2940
gctcgtgcct gtaatcccaa cactttggga ggccaaggca ggctgatcac ttgaagttgg 3000
gagttcaaga ccagcctggc caacatggtg aaatcccgtc tctactgaaa atacaaaaat 3060
taaccaggca tggtggtgtg tgcctgtagt cccaggaatc acttgaaccc aggaggcgga 3120
ggttgcagtg agctgagatc tcaccactgc acactgcact ccagcctggg caatggaatg 3180
agattccatc ccaaaaaata aaaaaataaa aaaataaaga acataccttg ggttgatcca 3240
cttaggaacc tcagataata acatctgcca cgtatagagc aattgctatg tcccaggcac 3300
tctactagac acttcataca gtttagaaaa tcagatgggt gtagatcaag gcaggagcag 3360
gaaccaaaaa gaaaggcata aacataagaa aaaaaatgga aggggtggaa acagagtaca 3420
ataacatgag taatttgatg ggggctatta tgaactgaga aatgaacttt gaaaagtatc 3480
ttggggccaa atcatgtaga ctcttgagtg atgtgttaag gaatgctatg agtgctgaga 3540
gggcatcaga agtccttgag agcctccaga gaaaggctct taaaaatgca gcgcaatctc 3600
cagtgacaga agatactgct agaaatctgc tagaaaaaaa acaaaaaagg catgtataga 3660
ggaattatga gggaaagata ccaagtcacg gtttattctt caaaatggag gtggcttgtt 3720
gggaaggtgg aagctcattt ggccagagtg gaaatggaat tgggagaaat cgatgaccaa 3780
atgtaaacac ttggtgcctg atatagcttg acaccaagtt agccccaagt gaaataccct 3840
ggcaatatta atgtgtcttt tcccgatatt cctcaggtac tccaaagatt caggtttact 3900
cacgtcatcc agcagagaat ggaaagtcaa atttcctgaa ttgctatgtg tctgggtttc 3960
atccatccga cattgaagtt gacttactga agaatggaga gagaattgaa aaagtggagc 4020
attcagactt gtctttcagc aaggactggt ctttctatct cttgtactac actgaattca 4080
cccccactga aaaagatgag tatgcctgcc gtgtgaacca tgtgactttg tcacagccca 4140
agatagttaa gtggggtaag tcttacattc ttttgtaagc tgctgaaagt tgtgtatgag 4200
tagtcatatc ataaagctgc tttgatataa aaaaggtcta tggccatact accctgaatg 4260
agtcccatcc catctgatat aaacaatctg catattggga ttgtcaggga atgttcttaa 4320
agatcagatt agtggcacct gctgagatac tgatgcacag catggtttct gaaccagtag 4380
tttccctgca gttgagcagg gagcagcagc agcacttgca caaatacata tacactctta 4440
acacttctta cctactggct tcctctagct tttgtggcag cttcaggtat atttagcact 4500
gaacgaacat ctcaagaagg tataggcctt tgtttgtaag tcctgctgtc ctagcatcct 4560
ataatcctgg acttctccag tactttctgg ctggattggt atctgaggct agtaggaagg 4620
gcttgttcct gctgggtagc tctaaacaat gtattcatgg gtaggaacag cagcctattc 4680
tgccagcctt atttctaacc attttagaca tttgttagta catggtattt taaaagtaaa 4740
acttaatgtc ttcctttttt ttctccactg tctttttcat agatcgagac atgggtggag 4800
gtggaagtgg tggaggtgga agtggtggag gtggaagtgg tggaggtgga agtggctccc 4860
actccatgag gtatttcagc gccgccgtgt cccggcccgg ccgcggggag ccccgcttca 4920
tcgccatggg ctacgtggac gacacgcagt tcgtgcggtt cgacagcgac tcggcgtgtc 4980
cgaggatgga gccgcgggcg ccgtgggtgg agcaggaggg gccggagtat tgggaagagg 5040
agacacggaa caccaaggcc cacgcacaga ctgacagaat gaacctgcag accctgcgcg 5100
gctactacaa ccagagcgag gccagttctc acaccctcca gtggatgatt ggctgcgacc 5160
tggggtccga cggacgcctc ctccgcgggt atgaacagta tgcctacgat ggcaaggatt 5220
acctcgccct gaacgaggac ctgcgctcct ggaccgcagc ggacactgcg gctcagatct 5280
ccaagcgcaa gtgtgaggcg gccaatgtgg ctgaacaaag gagagcctac ctggagggca 5340
cgtgcgtgga gtggctccac agatacctgg agaacgggaa ggagatgctg cagcgcgcgg 5400
acccccccaa gacacacgtg acccaccacc ctgtctttga ctatgaggcc accctgaggt 5460
gctgggccct gggcttctac cctgcggaga tcatactgac ctggcagcgg gatggggagg 5520
accagaccca ggacgtggag ctcgtggaga ccaggcctgc aggggatgga accttccaga 5580
agtgggcagc tgtggtggtg ccttctggag aggagcagag atacacgtgc catgtgcagc 5640
atgaggggct gccggagccc ctcatgctga gatggaagca gtcttccctg cccaccatcc 5700
ccatcatggg tatcgttgct ggcctggttg tccttgcagc tgtagtcact ggagctgcgg 5760
tcgctgctgt gctgtggaga aagaagagct cagattga 5798
<210> 10
<211> 1305
<212> DNA/RNA
<213> Artificial Sequence
<220>
<221> gene
<222> (1)..(1305)
<223> nucleic acid sequence encoding B2M-HLA-G5 fusion protein
<400> 10
atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctctttctgg cctggaggct 60
atccagcgta ctccaaagat tcaggtttac tcacgtcatc cagcagagaa tggaaagtca 120
aatttcctga attgctatgt gtctgggttt catccatccg acattgaagt tgacttactg 180
aagaatggag agagaattga aaaagtggag cattcagact tgtctttcag caaggactgg 240
tctttctatc tcttgtacta cactgaattc acccccactg aaaaagatga gtatgcctgc 300
cgtgtgaacc atgtgacttt gtcacagccc aagatagtta agtgggatcg agacatgggt 360
ggaggtggaa gtggtggagg tggaagtggt ggaggtggaa gtggtggagg tggaagtggc 420
tcccactcca tgaggtattt cagcgccgcc gtgtcccggc ccggccgcgg ggagccccgc 480
ttcatcgcca tgggctacgt ggacgacacg cagttcgtgc ggttcgacag cgactcggcg 540
tgtccgagga tggagccgcg ggcgccgtgg gtggagcagg aggggccgga gtattgggaa 600
gaggagacac ggaacaccaa ggcccacgca cagactgaca gaatgaacct gcagaccctg 660
cgcggctact acaaccagag cgaggccagt tctcacaccc tccagtggat gattggctgc 720
gacctggggt ccgacggacg cctcctccgc gggtatgaac agtatgccta cgatggcaag 780
gattacctcg ccctgaacga ggacctgcgc tcctggaccg cagcggacac tgcggctcag 840
atctccaagc gcaagtgtga ggcggccaat gtggctgaac aaaggagagc ctacctggag 900
ggcacgtgcg tggagtggct ccacagatac ctggagaacg ggaaggagat gctgcagcgc 960
gcggaccccc ccaagacaca cgtgacccac caccctgtct ttgactatga ggccaccctg 1020
aggtgctggg ccctgggctt ctaccctgcg gagatcatac tgacctggca gcgggatggg 1080
gaggaccaga cccaggacgt ggagctcgtg gagaccaggc ctgcagggga tggaaccttc 1140
cagaagtggg cagctgtggt ggtgccttct ggagaggagc agagatacac gtgccatgtg 1200
cagcatgagg ggctgccgga gcccctcatg ctgagatgga gtaaggaggg agatggaggc 1260
atcatgtctg ttagggaaag caggagcctc tctgaagacc tttaa 1305
<210> 11
<211> 19
<212> DNA/RNA
<213> Artificial Sequence
<220>
<221> misc_RNA
<222> (1)..(19)
<223> gRNA sequence targeting CIITA site
<400> 11
gggaggctta tgccaatat 19

Claims (12)

1. A method of preparing an immunocompatible human pluripotent stem cell comprising engineering the human pluripotent stem cell to:
(a) Free B2M protein is not expressed, HLA-G1 and secretory HLA-G5 are expressed; and
(b) The CIITA protein is not expressed.
2. The method of claim 1, wherein in (a),
modifying the genome of the human pluripotent stem cell, fusing the polynucleotide encoding HLA-G1 with an endogenous B2M gene in the human pluripotent stem cell, thereby expressing a B2M-HLA-G1 fusion protein and not expressing a free B2M protein; preferably, the HLA-G1 encoding gene is introduced into the endogenous B2M gene at a position before the stop codon in exon 3, or the stop codon in exon 3 of the endogenous B2M gene is replaced with the HLA-G1 encoding gene; more preferably, the modification is performed by a gene editing method; and
Introducing exogenous polynucleotide encoding a B2M-HLA-G5 fusion protein into a human pluripotent stem cell, the B2M-HLA-G5 fusion protein comprising B2M and HLA-G5; preferably, the exogenous polynucleotide encoding the B2M-HLA-G5 fusion protein is introduced using a recombinant vector; more preferably, the recombinant vector is a viral vector, preferably a lentiviral vector.
3. The method of claim 2, wherein,
the B2M-HLA-G1 fusion protein also comprises a flexible connecting peptide between B2M and HLA-G1, preferably a flexible connecting peptide shown as SEQ ID NO. 3; and/or, the B2M-HLA-G1 fusion protein comprises the following components from the N end to the C end in sequence: B2M and HLA-G1; preferably, the amino acid sequence of the B2M-HLA-G1 fusion protein is shown as SEQ ID NO. 4; more preferably, the nucleic acid sequence of the B2M-HLA-G1 fusion protein is shown as SEQ ID NO. 9; and/or
The B2M-HLA-G5 fusion protein also comprises a flexible connecting peptide between B2M and HLA-G5, preferably a flexible connecting peptide shown as SEQ ID NO. 3; and/or, the B2M-HLA-G5 fusion protein comprises the following components from the N end to the C end in sequence: B2M and HLA-G5; preferably, the amino acid sequence of the B2M-HLA-G5 fusion protein is shown as SEQ ID NO. 8; more preferably, the nucleic acid sequence of the B2M-HLA-G5 fusion protein is shown as SEQ ID NO. 10.
4. The method of claim 1, wherein in (b), the knockout is made for exon 3 of the CIITA gene in the human pluripotent stem cell genome; preferably, the knockout is performed by a gene editing method; more preferably, the gene editing is performed with the gRNA of the nucleotide sequence shown in SEQ ID NO. 11.
5. The method of any one of claims 1 to 4, comprising one or more of the following a-D:
A. the HLA-G1 comprises: a polypeptide with an amino acid sequence shown as SEQ ID NO. 1; or, a derivative polypeptide having an amino acid sequence having 90% or more sequence identity to SEQ ID NO. 1 and having the function of the polypeptide shown in SEQ ID NO. 1;
B. the B2M includes: a polypeptide with an amino acid sequence shown as SEQ ID NO. 2; or, a derivative polypeptide having an amino acid sequence having 90% or more sequence identity to SEQ ID NO. 2 and having the function of the polypeptide represented by SEQ ID NO. 2;
C. the CIITA protein comprises: a polypeptide with an amino acid sequence shown as SEQ ID NO. 5; or, a derivative polypeptide having an amino acid sequence having 90% or more sequence identity to SEQ ID NO. 5 and having the function of the polypeptide shown in SEQ ID NO. 5;
D. the HLA-G5 comprises: a polypeptide with an amino acid sequence shown as SEQ ID NO. 6; or a polypeptide fragment having an amino acid sequence having 90% or more sequence identity to SEQ ID NO. 6 and having the function of the polypeptide shown in SEQ ID NO. 6.
6. An immunocompatible human pluripotent stem cell that (a) does not express free B2M protein, expresses HLA-G1 and secretory HLA-G5; and (b) does not express CIITA protein;
preferably, the human pluripotent stem cells: the endogenous B2M gene in the genome is fused with a polynucleotide encoding HLA-G1; comprising an exogenous polynucleotide encoding a B2M-HLA-G5 fusion protein; and, the CIITA gene is knocked out in the genome.
7. The immunocompatible human pluripotent stem cell according to claim 6, wherein the cell is constructed by the method according to any one of claims 1 to 5.
8. Use of the immunocompatible human pluripotent stem cells of claim 6 or 7 for preparing cells suitable for transplantation by induced differentiation;
preferably, the cells suitable for transplantation are tissue or organ cells;
more preferably, the tissue or organ cells comprise: cardiovascular precursor cells, cardiomyocytes, endothelial cells, smooth muscle cells, nerve cells, hematopoietic stem cells, myeloid cells, gonococcal cells, retinal pigment epithelial cells, islet B cells, liver cells, keratinocytes, skeletal muscle cells, adipocytes, bone cells, chondrocytes, mesenchymal stem cells;
More preferably, wherein said myeloid cells comprise granulocytes, monocytes, macrophages, erythrocytes, platelets, and/or wherein said gonococcal cells comprise natural killer cells, T cells, B cells, and/or wherein said hepatic cells comprise hepatocytes, cholangiocytes, hepatic endothelial cells, hepatic stellate cells, kupffer cells, mesothelial cells.
9. A method of preparing cells suitable for transplantation, comprising: (a) Preparing an immunocompatible human pluripotent stem cell by the method of any one of claims 1 to 5; (b) Further inducing differentiation of the cells of (a) to obtain cells suitable for transplantation;
preferably, the cells suitable for transplantation are tissue or organ cells; more preferably, the tissue or organ cells comprise: cardiovascular precursor cells, cardiomyocytes, endothelial cells, smooth muscle cells, nerve cells, hematopoietic stem cells, myeloid cells, gonococcal cells, retinal pigment epithelial cells, islet B cells, liver cells, keratinocytes, skeletal muscle cells, adipocytes, bone cells, chondrocytes, mesenchymal stem cells;
more preferably, wherein said myeloid cells comprise granulocytes, monocytes, macrophages, erythrocytes, platelets, and/or wherein said gonococcal cells comprise natural killer cells, T cells, B cells, and/or wherein said hepatic cells comprise hepatocytes, cholangiocytes, hepatic endothelial cells, hepatic stellate cells, kupffer cells, mesothelial cells.
10. The method of claim 9, wherein the tissue or organ cells are cardiomyocytes and are prepared by CHIR99021 followed by IWR-1 induction; or (b)
The tissue or organ cells are endothelial cells and are prepared by adopting a method of induction of CHIR99021, bFGF and VEGF+BMP4 in sequence.
11. A cardiomyocyte or endothelial cell derived from an immune-compatible human pluripotent stem cell prepared by the method according to any of claims 1 to 5 or the immune-compatible human pluripotent stem cell according to claim 6 or 7.
12. Use of a cardiomyocyte according to claim 11 in the manufacture of a composition or medicament for treating a heart-related disease or disorder; preferably, the heart-related disease or disorder is myocardial injury, myocardial infarction, myocardial ischemia, ischemia reperfusion injury, or other heart injury; more preferably, the heart-related disease or disorder is ischemia/reperfusion injury.
CN202210752354.6A 2022-06-29 2022-06-29 Immune compatible human pluripotent stem cell, preparation method and application thereof Pending CN117343962A (en)

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